EP3328377A1 - Compositions et méthodes pour thérapies immuno-oncologiques - Google Patents

Compositions et méthodes pour thérapies immuno-oncologiques

Info

Publication number
EP3328377A1
EP3328377A1 EP16833628.7A EP16833628A EP3328377A1 EP 3328377 A1 EP3328377 A1 EP 3328377A1 EP 16833628 A EP16833628 A EP 16833628A EP 3328377 A1 EP3328377 A1 EP 3328377A1
Authority
EP
European Patent Office
Prior art keywords
conjugate
cell
antigen
tumor
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16833628.7A
Other languages
German (de)
English (en)
Other versions
EP3328377A4 (fr
Inventor
Sudhakar Kadiyala
Donna T. Ward
Richard Wooster
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tarveda Therapeutics Inc
Original Assignee
Tarveda Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tarveda Therapeutics Inc filed Critical Tarveda Therapeutics Inc
Publication of EP3328377A1 publication Critical patent/EP3328377A1/fr
Publication of EP3328377A4 publication Critical patent/EP3328377A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of immuno-oncology therapy.
  • the present invention relates to immune modulating conjugates and particles packaging such conjugates.
  • Cancer is a heterogeneous disease that results from a multi-step process, characterized by uncontrolled tumor cell proliferation, invasion and metastasis. Tumor cells have also the ability to evade cell death and to escape immune system surveillance (Zitvogel et al, Nat. Rev. Immunol. 2006, 6:715-727). With a more detailed understanding of the interaction between the immune system and cancer, cancer immunotherapy has become a promising therapeutic strategy for treatment of cancer.
  • the immune system can recognize tumor cells, parts of tumor cells or specific substances isolated from tumor cells and respond to these malignant cells. Both the innate and adaptive immune subsystems can respond to tumor cells in vivo. In the adaptive immune response, antigen presenting cells (e.g., dendritic cells) can capture and present tumor specific antigens to naive T cells producing activated T-cells. Activated cancer antigen specific T cells can recognize and destroy tumor cells presenting epitopes to which the T cells have been primed. The ability to exploit the immune system has brought new insights into the development of novel cancer immunotherapy treatments.
  • antigen presenting cells e.g., dendritic cells
  • Activated cancer antigen specific T cells can recognize and destroy tumor cells presenting epitopes to which the T cells have been primed.
  • adoptive T cell immunotherapy has had impressive success in treating malignant and infection diseases.
  • Autologous T cells are cultured and/or engineered ex vivo and adoptively transferred into the patient.
  • T cells are directly targeted in vivo by vaccination or biological compounds.
  • these immunotherapies generate a de novo T cell-mediated immune response and/or enhance preexisting functions, which are often suppressed in patients (Reviewed by Perica et al, Adoptive T cell immunotherapy, 2015, Rambam Maimonides Med J . 6(1): e0004).
  • Cancer immunotherapies may be suitable for a large number of cancer types.
  • the present invention providesnovel conjugates and nanoparticles for targeted immunotherapy.
  • the conjugates and nanoparticles described here in can increase the delivery of immunologic agents such as tumor specific antigens, artificial antigen presenting cells, T cell agents that can activate T cells, antibodies, cytokines and other immune stimulating agents to a targeted tissue (e.g., a metastatic site, or a lymph node), and/ or a particular cell type of interest such as tumor cells and a type of immune cells.
  • the conjugates and nanoparticles provide flexibility for combining different agents that function for different mechanisms to the same conjugate or the same particle; such combinations may
  • conjugates and nanoparticles are useful in the sustained release of immunologic active agents.
  • the present invention provides compositions for cancer immunotherapyand builds upon previous work by Bilodeau et. al, in WO2014/106208, the contents which are incorporated by reference in their entirety.
  • the compositions include conjugates and nanoparticles, useful for the production of cancer vaccines, and activated T cells for adoptive cellular immunotherapy.
  • the conjugates of the present invention are constructed to compromise a targeting moiety, a linker and an active agent.
  • the targeting moiety may specifically target to a tumor cell or an immune cell.
  • the active agent may comprise any agent that can manipulate cancer-specific immune responses positively, such as tumor associated antigens, agents that can enhance antigen presentation by antigen presenting cells such as dendritic cells, agents that can stimulate activation of cancer specific T cells, antibodies, and cytokines, chemokines and other immunoregulatory molecules.
  • agent that can manipulate cancer-specific immune responses positively such as tumor associated antigens, agents that can enhance antigen presentation by antigen presenting cells such as dendritic cells, agents that can stimulate activation of cancer specific T cells, antibodies, and cytokines, chemokines and other immunoregulatory molecules.
  • a variety of strategies have been developed to elicit cancer specific immune responses. These strategies are developed to increase tumor antigen presentation by antigen presenting cells (APCs, e.g., dendritic cells), or to enhance cancer specific T cell
  • APCs antigen presenting cells
  • dendritic cells dendritic cells
  • the present conjugates provide platforms for cancer immunotherapy modalities.
  • the conjugate comprise three moieties: an active agent, a targeting moiety and a linker that connects the active agent and the targeting moiety.
  • the active agent may be an agent that can stimulate/increase a cancer specific immune response.
  • agents include antibodies specific to a tumor antigen; tumor antigenic peptides (i.e., epitopes) that can increase the antigen presentation to T cells; agents that can stimulate proliferation (e.g., cytokines), expansion, maturation and migration of antigen presenting cells (e.g., dendritic cells), and/or increase antigen capture and processing in antigen presenting cells; agents that enhance cancer specific T cells expansion, proliferation and migration, and/or increase antigen recognition; cytokines and chemokines that positively regulate immune responses; or agents that can inhibit immunosuppressive signals in the tumor tissues.
  • tumor antigenic peptides i.e., epitopes
  • agents that can stimulate proliferation e.g., cytokines
  • expansion, maturation and migration of antigen presenting cells e.g., dendritic cells
  • agents that enhance cancer specific T cells expansion, proliferation and migration, and/or increase antigen recognition cytokines and chemokines that positively regulate immune responses; or agents that can inhibit immunosuppressive
  • the targeting moiety of the conjugate can function to deliver an active agent of the conjugate to a targeted area such as a tumor tissue or lymph node, or a type of cell of interest such as T cells, dendritic cells and/or NK cells.
  • the targeting moiety itself may have an immune stimulating activity, the same or different from the active agent in the same conjugate.
  • the linker of the conjugate not only connects the active moiety and the targeting moiety, in some cases, but may also control/assist in the release of the active agent to a targeted area or a cell. It some aspects, it may provide a sustained release of the active agent for a period of time.
  • Design of the present conjugates is flexible and may be configured in various combinations depending on types, origins, metastatic status, and other clinical and pathological status of the cancers to be related.
  • one or more active agents from the same category such as different tumor antigen peptides from one common tumor associated antigen protein, or from different tumor associated antigen proteins but associated with one type of tumor; or from a combination of tumor associated antigens isolated from a single patient, i.e. personalized, may be connected to a targeting moiety through the linker in a conjugate.
  • active agents may function through different mechanisms. Two or more active agent such as tumor specific antigenic epitopes and agents for increasing dendritic cell antigen capture may be connected in one conjugate. In another example, one or more tumor specific antigenic peptide, and one or more immune costimulatory molecule agonists may be included in one conjugate to increase the efficacy of tumor specific T cell activation.
  • more than one targeting moiety may be linked to active agents of the conjugate for targeting different tissues, cells or even different intracellular components such as those of the cell surface and cytoplasm.
  • the present invention also provide particles, nanoparticles and/or polymeric nanoparticles that can encapsulate one or more conjugates of the present invention, providing an improved nanodelivery system.
  • the present nano-delivery system improves pharmacokinetics, targeting of tissues and cells to enhance efficacy, specificity and lower toxicity.
  • the present conjugates designed for increasing immune response, and particles comprising such conjugates provide more specific compositions and methods to fight cancer.
  • APCs such as macrophages are good at phagocytosis and may be stimulated by nanoparticles. The active agents of the conjugates in the nanoparticle are then released inside the APCs.
  • the active agents are only released within certain environments, such as with the presence of lysozymes.
  • particles, nanoparticles and/or polymeric nanoparticles target bone marrow and delivers conjugates to bone marrow.
  • Such solid nanoparticles and their preparation are taught in, for example, WO2014/106208, the contents of which are incorporated herein in their entirety.
  • Administration means the actual physical introduction of the composition into or onto (as appropriate) the host. Any and all methods of introducing the composition into the host are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein
  • Adoptive cellular immunotherapy As used herein, the terms “adoptive cellular immunotherapy ' " or “adoptive immunotherapy ' or “T cell immunotherapy ' “ , or “Adoptive T cell therapy (ACT)”, are used interchangeably.
  • Adoptive immunotherapy uses T cells that a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. The injection of a large number of activated tumor specific T cells can induce complete and durable regression of cancers.
  • agonist refers to any substance that binds to a target (e.g. a receptor); and activates or increases the biological activity of the target.
  • a target e.g. a receptor
  • an “agonist” antibody is an antibody that activates or increases the biological activity of the antigen(s) it binds.
  • Antagonist refers to any agent that inhibits or reduces the biological activity of the target(s) it binds.
  • an “antagonist” antibody is an antibody that inhibits or reduces biological activity of the antigen(s) it binds.
  • Antigen As used herein, the terms “antigen "or "immunogen,” as being used interchangeably, is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itself. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells such as cytotoxic T lymphocytes and T helper cells, or both.
  • An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates.
  • antigenic or “immunogenic” refers to a structure that is an antigen. These terms are used interchangeably.
  • Antigen presenting cells refers to cells that process antigens and present peptide epitopes on the cell surface via MHC molecules; APCs include dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells. Dendritic cells (DCs) and macrophages are antigen presenting cells in vivo. The dendritic cells are more efficient APCs than macrophages. These cells are usually found in structural compartments of the lymphoid organs such as the thymus, lymph nodes and spleen, and in the bloodstream and other tissues of the body as well.
  • Antibodies are specialized proteins called
  • immunoglobulins that specifically recognize and bind to specific antigens that caused their stimulation. Antibody production by B lymphocytes in vivo and binding to foreign antigens is often critical as a means of signaling other cells to engulf, kill or remove that substance that contains the foreign antigens from the body.
  • An immunoglobulin is a protein comprising one or more polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Also subclasses of the heavy chain are known. For example, IgG heavy chains in humans can be any of IgGl, IgG2, IgG3 and IgG4 subclass.
  • Antibodies may exist as full length intact antibodies or as a number of well- characterized fragments produced by digestion with various peptidases or chemicals, such as F(ab')2, a dimer of Fab which itself is a light chainjoined to VH-CHl by a disulfide bond; an Fab' monomer, a Fab fragment with the hinge region; and a Fc fragment, a portion of the constant region of an immunoglobulin.
  • F(ab')2 a dimer of Fab which itself is a light chainjoined to VH-CHl by a disulfide bond
  • an Fab' monomer a Fab fragment with the hinge region
  • Fc fragment a portion of the constant region of an immunoglobulin.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that any of a variety of antibody fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • the term antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo or antibodies and fragments obtained by using recombinant DNA methodologies.
  • Recombinant antibodies may be conventional full length antibodies, antibody fragments known from proteolytic digestion, unique antibody fragments such as Fv or single chain Fv (scFv), domain deleted antibodies, and the like.
  • An Fv antibody is about 50 Kd in size and comprises the variable regions of the light and heavy chain.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH: :VL heterodimer.
  • An antibody may be a non-human antibody, a human antibody, a humanized antibody or a chimeric antibody.
  • the "chimeric antibody” means a genetically engineered fusion of parts of a non-human (e.g., mouse) antibody with parts of a human antibody.
  • chimeric antibodies contain approximately 33% non-human protein and 67% human protein. Developed to reduce the HAMA response elicited by non-human antibodies, they combine the specificity of the non-human antibody with the efficient human immune system interaction of a human antibody.
  • a human antibody may be a "fully human” antibody.
  • the terms "human” and 'fully human” is used to label those antibodies derived from transgenic mice carrying human antibody genes or from human cells. To the human immune system, however, the difference between "fully human” “humanized”, and “chimeric” antibodies may be negligible or nonexistent and as such all three may be of equal efficacy and safety.
  • Autologous As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
  • Cancer refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and grow results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • Combination therapy means a therapy strategy that embraces the administration of therapeutic compositions of the present invention (e.g., conjugates comprising one or more neoantigens) and one or more additional therapeutic agents as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected). In combination therapy, combined therapeutic agent may be administered in a sequential manner, or by substantially simultaneous administration.
  • Compound As used herein, the term “"compound, " as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. In the present application, compound is used interchangeably with conjugate. Therefore, conjugate, as used herein, is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1 ,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds.
  • “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes of hydrogen include tritium and deuterium.
  • the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • Copolymer generally refers to a single polymeric material that is comprised of two or more different monomers.
  • the copolymer can be of any form, such as random, block, graft, etc.
  • the copolymers can have any end-group, including capped or acid end groups.
  • Cytokine As used herein, the term “cytokine” refers to a substance secreted by certain cells of the immune system and has a biological effect on other cells. Cytokines can be a number of different substances such as interferons, interleukins and growth factors.
  • Cytotoxic agent means a substance that inhibits or prevents the function of cells and/or causes destruction of cells, such as radioactive isotopes, chemotherapeutic agents, and toxins.
  • Cytotoxic T cell As used herein, the terms “cytotoxic T cell (TC)” or “cytotoxic T lymphocyte (CTL)”, or “T-killer cells”, or “CD8+ T-cell” or “killer T cell” are used interchangeably.
  • T lymphocytes that can recognize abnormal cells including cancer cells, cells that are infected particularly by viruses, and cells that are damaged in other ways and induce the death of such cells.
  • Epitope As used herein, the term “epitope” means a small peptide structure formed by contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and about 9, or about 8-15 amino acids.
  • a T cell epitope means a peptide which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by native T cells, cytotoxic T- lymphocytes or T-helper cells, respectively.
  • HLA Human Leukocyte Antigen
  • HLA proteins Human Leokocyte Antigens
  • MHC Major Histocompatibility Complex
  • MHC molecules MHC proteins
  • MHC proteins proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells.
  • the major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and/or foreign antigens and thus for regulating immunological processes.
  • the major histocompatibility complex is classified into two gene groups coding for different proteins, namely molecules of MHC class I and molecules of MHC class II.
  • the molecules of the two MHC classes are specialized for different antigen sources.
  • the molecules of MHC class I present endogenously synthesized antigens, for example viral proteins and tumor antigens.
  • the molecules of MHC class II present protein antigens originating from exogenous sources, for example bacterial products.
  • the cellular biology and the expression patterns of the two MHC classes are adapted to these different roles.
  • MHC class I molecules (called HLA class I in human) consist of a heavy chain and a light chain and are capable of binding a short peptide with suitable binding motifs, and presenting it to cytotoxic T-lymphocytes.
  • the peptide bound by the MHC molecules of class I originates from an endogenous protein antigen.
  • the heavy chain of the MHC molecules of class I is preferably an HLA- A, HLA-B or HLA-C monomer, and the light chain is ⁇ -2- microglobulin.
  • MHC class II molecules (called HLA class II in human) consist of an a-chain and a ⁇ -chain and are capable of binding a short peptide with suitable binding motifs, and presenting it to T-helper cells.
  • the peptide bound by the MHC molecules of class II usually originates from an extracellular of exogenous protein antigen.
  • the a-chain and the ⁇ -chain are in particular HLA-DR, HLA-DQ, HLA-DP, HLA-DO and HLA-DM monomers.
  • Immune cell refers to a cell that is capable of participating, directly or indirectly, in an immune response.
  • Immune cells include, but are not limited to T-cells, B-cells, antigen presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhan's cells, stem cells, peripheral blood mononuclear cells, cytotoxic T-cells, tumor infiltrating lymphocytes (TIL), etc.
  • TIL tumor infiltrating lymphocytes
  • APC antigen presenting cell
  • DC dendritic cell
  • Dendritic cell or “DC” refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC- restricted T cells and are very effective at presenting antigens to T cells in situ.
  • the antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes.
  • an "activated DC” is a DC that has been pulsed with an antigen and capable of activating an immune cell.
  • T- cell as used herein, is defined as a thymus-derived cell that participates in a variety of cell- mediated immune reactions, including CD8+ T cell and CD4+ T cell.
  • B-cell as used herein, is defined as a cell derived from the bone marrow and/or spleen. B cells can develop into plasma cells which produce antibodies.
  • Immune response means a defensive response a body develops against "foreigner” such as bacteria, viruses and substances that appear foreign and harmful.
  • An anti-cancer immune response refers to an immune surveillance mechanism by which a body recognizes abnormal tumor cells and initiates both the innate and adaptive of the immune system to eliminate dangerous cancer cells.
  • the innate immune system is a non-specific immune system that comprises the cells (e.g., Natural killer cells, mast cells, eosinophils, basophils; and the phagocytic cells including macrophages, neutrophils, and dendritic cells) and mechanisms that defend the host from infection by other organisms.
  • An innate immune response can initiate the productions of cytokines, and active complement cascade and adaptive immune response.
  • the adaptive immune system is specific immune system that is required and involved in highly specialized systemic cell activation and processes, such as antigen presentation by an antigen presenting cell; antigen specific T cell activation and cytotoxic effect.
  • Linker refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long.
  • heteroatoms e.g., nitrogen, oxygen, sulfur, etc.
  • Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted.
  • linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • Linkers may include any of those taught in, for example, WO2014/10628, the contents of which are incorporated herein by reference in their entirety.
  • mean particle size generally refers to the statistical mean particle size (diameter) of the particles in the composition.
  • the diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer to the hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle.
  • Mean particle size can be measured using methods known in the art such as dynamic light scattering. Two populations can be said to have a "substantially equivalent mean particle size" when the statistical mean particle size of the first population of particles is within 20% of the statistical mean particle size of the second population of particles; for example, within 15%, or within 10%.
  • monodisperse and “homogeneous size distribution, " as used interchangeably herein, describe a population of particles, microparticles, or nanoparticles all having the same or nearly the same size.
  • a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.
  • Peptide refers to a molecule composed of a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. Peptide sometimes is used interchangeably with the term "polypeptide.” Polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described. In some embodiments, peptides are less than 50 amino acids in length.
  • Targeting moiety refers to a moiety that binds to or localizes to a specific locale.
  • the moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule.
  • the locale may be a tissue, a particular cell type, or a subcellular compartment.
  • a targeting moiety can specifically bind to a selected component of the targeted locale.
  • Tumor associated antigen As used herein, the term “tumor associated antigen (TAA)” refers to an antigenic substance produced in tumor cells. Tumor associated antigens may be encoded by a primary open reading frame of gene products that are differentially expressed by tumors, and not by normal tissues. They may also be encoded by mutated genes, intronic sequences, or translated alternative open reading frames,
  • pseudogenes represent the products of gene translocation events.
  • Tumor-associated antigens can derive from any protein or glycoprotein synthesized by the tumor cell.
  • TAA proteins can reside in any subcellular compartment of the tumor cell; i.e., they may be membrane-bound, cytoplasmic, nuclear-localized, or even secreted by the tumor cells.
  • a TAA may allow for a preferential recognition of tumor cells by specific T cells or immunoglobulins, therefore activate an anti-tumor immune response to kill tumor cells.
  • Vaccine refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases.
  • compositions of the present inventions include conjugates comprising a targeting moiety, a linker, and one or more active agents, e.g., one or more immuno-oncological agents conjugated to the targeting moiety through a linker.
  • Nanoparticles that package one or more such conjugates are also provided.
  • the conjugates can be encapsulated into nanoparticles or disposed on the surface of the particles.
  • conjugates of the present invention and nanoparticles comprising such conjugates may be used as immuno-oncological agents such as cancer vaccines, or as adjuvants to enhance anti-cancer immune responses in combination with other immunotherapies, or to generate cancer vaccines in vitro for in vivo cellular immunotherapy.
  • the conjugates, nanoparticles comprising the conjugates, and/or formulations thereof can provide improved temporospatial delivery of the active agent and/or improved biodistribution compared to delivery of the active agent alone.
  • Conjugates, nanoparticles and other compositions of the present invention provide a system that is flexible in tailoring the composition and numbers of active agents (e.g., flexible addition and subtraction of active agents connected to the targeting moiety) important for harnessing an anti-tumor immune response, for example, antigen specific T cell activation and response.
  • Conjugates, nanoparticles and other compositions of the present invention may provide increased targeting properties since the targeting moieties of the conjugates specifically target to a selected tissue and/or certain types of cells of interest.
  • Conjugates, nanoparticles and other compositions of the present invention may coordinate action of the innate and adaptive phases of the immune system to produce an effective anti-cancer immune response. In some instances, they may be used for active immunotherapy and adoptive immunotherapy of cancer and/or other diseases (e.g., viral infection).
  • diseases e.g., viral infection.
  • conjugates, nanoparticles and other compositions comprising conjugates may include a B cell immune response in subject.
  • conjugates, nanoparticles and other compositions comprising conjugates may include a CD4+ T cell immune response in a subject.
  • conjugates, nanoparticles and other compositions comprising conjugates may induce a CD8+ T cell immune response in a subject.
  • conjugates, nanoparticles and other compositions of the present invention may also be used for in vivo and ex vivo activation and expansion of lymphocytes including T cells to elicit an anti-tumor immune response.
  • conjugates comprise at least three moieties: a targeting moiety (or ligand), a linker, and an active agent called a payload that is connected to the targeting moiety via the linker.
  • the conjugate may be a conjugate between a single active agent and a single targeting moiety with the formula: X- Y-Z, wherein X is the targeting moiety; Y is a linker; and Z is the active agent.
  • One targeting moiety can be conjugated to two or more payloads wherein the conjugate has the formula: X-(Y-Z) n .
  • one active payload can be linked to two or more targeting ligands wherein the conjugate has the formula: (X-Y)n-Z.
  • one or more targeting ligands may be connected to one or more active payloads wherein the conjugate formula may be (X-Y-Z) n .
  • the formula of the conjugates maybe, for example, X-Y-Z-Y-X, (X-Y-Z)n-Y-Z, or X-Y-(X-Y- Z) n , wherein X is a targeting moiety; Y is a linker; Z is an active agent.
  • each moiety in the conjugate may vary dependent on types of agents, sizes of the conjugate, delivery targets, particles used to packaging the conjugate, other active agents (e.g., immunologic adjuvants) and routes of administration.
  • Each occurrence of X, Y, and Z can be the same or different, e.g. the conjugate can contain more than one type of targeting moiety, more than one type of linker, and/or more than one type of active agent, n is an integer equal to or greater than 1. In some embodiments, n is an integer between 1 and 50, or between 2 and 20, or between 5 and 40. In some embodiments, n may be an integer of 2, 3, 4, 5, 6, 7, 8.
  • the conjugate may comprise pendent or terminal functional groups that allow further modification or conjugation.
  • the pendent or terminal functional groups may be protected with any suitable protecting groups.
  • Conjugates of the present invention may target discrete pathways involved in critical processes of anti-cancer immune responses. These critical processes may include antigen degradation and processing, activation of dendritic cells to present antigenic epitopes, production of cytokines (e.g., interferons), expression of co-stimulatory ligands, induction of a productive T cell response for example within lymph nodes, migration of activated T cells to the tumor microenvironment in response to chemokines and homing receptor expression, having effector T cells (e.g., CD4+ T cells and CD8+ T cells) gain access to antigen expressing tumor cells and maintenance of sufficient functionality of effector T cell to destroy tumor cells.
  • cytokines e.g., interferons
  • co-stimulatory ligands e.g., induction of a productive T cell response for example within lymph nodes
  • effector T cells e.g., CD4+ T cells and CD8+ T cells
  • cancer antigens as payloads of the conjugates, may be delivered to antigen presenting cells (APCs) (e.g., dendritic cells) through a targeting moiety with increased targeting delivery, therefore, enhancing the immunogenicity of TAAs to induce TAA specific cytotoxic T-lymphocytes (CTL).
  • APCs antigen presenting cells
  • CTL cytotoxic T-lymphocytes
  • the conjugate comprises a payload that binds to a chimeric antigen receptor (CAR) T cell, a linker, and a targeting moiety that binds to a tumor cell.
  • the targeting moiety may bind to a cell surface protein on tumor cells, such as but not limited to a folate receptor, a somatostatin receptor (SSTR), or a luteinizing hormone-releasing hormone receptor (LHRHR).
  • the payload may be a single chain variable fragment (scFV) that binds to a cell surface protein on CAR T cells.
  • scFV single chain variable fragment
  • TAAs Tumor associated antigens
  • Payload may be any active agents such as therapeutic agents, prophylactic agents, or diagnostic /prognostic agents.
  • a payload may have a capability of manipulating a physiological function (e.g., anti-cancer immune response) in a subject.
  • a physiological function e.g., anti-cancer immune response
  • One or more, either the same or different payloads may be included in the present conjugate.
  • a payload may be an active agent that can boost or provoke an anti-cancer immune response in a subject.
  • Immunotherapy is an advantageous strategy to treat cancer. Any compound that can provoke and/or enhance an immune response to destroy tumor cells in a subject may be included in the present conjugate.
  • agents may be tumor associated antigens (TAAs), antigen epitopes including antigen peptides presented by either MHC (major histocompatibility complex) class I or MHC class II molecules; cytokines, chemokines, other immunomodulators, T cell receptors (TCRs), CD (cell differentiation molecules) antigens, antibodies, cytotoxic agents, cell adhesion molecules and any components that are involved in an immune response; or variants thereof.
  • a payload may be a protein including a peptide, a nucleic acid, a sugar, a lipid, a lipoprotein, a glycoprotein, a glycolipid, or a small molecule.
  • the plural payloads may belong to the same category such as multiple epitope peptides derived from a single TAA, or multiple different tumor associated antigens isolated from a tumor tissue.
  • a plural of payloads having different functionality such as a mix of tumor associated antigens and co-stimulatory factors may be included in the same conjugate to synergistically enhance the antigen presentation to T cells.
  • TAAs tumor associated antigens
  • epitope peptides derived from TAAs can be selected as antigens to selectively stimulate cytotoxic T lymphocyte (CTL) response.
  • CTL cytotoxic T lymphocyte
  • MHC/HLA molecules used for presenting antigens.
  • MHC/HLA class I molecules are expressed on the surface of all cells and MHC/HLA class II are expressed on the surface of professional antigen presenting cells (APCs).
  • APCs professional antigen presenting cells
  • MHC/HLA class II molecules bind primarily to peptides derived from proteins made outside of an APC, but can present self (endogenous) antigens.
  • HLA class I molecules bind to peptides derived from proteins made inside a cell, including proteins expressed by an infectious agent (e.g., such as a virus) in the cell and by a tumor cell.
  • HLA class I proteins When the HLA class I proteins reach the surface of the cell these molecules will thus display any one of many peptides derived from the cytosolic proteins of that cell, along with normal "self peptides being synthesized by the cell. Peptides presented in this way are recognized by T-cell receptors which engage T-lymphocytes in an immune response against the antigens to induce antigen-specific cellular immunity.
  • a payload may be a TAA or an antigenic peptide (epitope) derived from a TAA.
  • An antigenic peptide may be a CD8 + T cell epitope that binds to specific MHC (HLA in human) class I molecules with a high affinity.
  • An antigenic peptide may be a CD4+ T cell epitope that binds to specific MHC (HLA in human) class II molecules with a high affinity.
  • the antigenic peptide may be about 5 to 50 amino acids in length.
  • the antigenic peptide may be greater than 5 amino acids in length, or greater than 10 amino acids in length, or greater than 15 amino acids in length, or greater than 20 amino acids in length, or greater than 25 amino acids in length, or greater than 30 amino acids in length, or greater than 35 amino acids in length, or greater than 40 amino acids in length, or greater than 45 amino acids in length.
  • the antigenic peptide may contain 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids.
  • the antigenic peptide be as small as possible while still maintaining substantially all of the immunologic activity of the native protein.
  • the HLA class I binding antigenic peptides may have a length of about 6 to about 15 amino acid residues, for example, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15.
  • the HLA class II binding peptides may have about 6 to about 30 amino acid residues, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, preferably to between about 13 and about 20 amino acids, e.g., 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
  • the antigenic epitope from a TAA may be an epitope that induces a B cell response in a subject to generate TAA specific antibody mediated immune responses.
  • TAAs or TAA derived antigenic peptides may be delivered directly to activate T cells through the targeting moieties of the conjugate.
  • Conjugates of the present invention comprising one or more TAAs and/or antigenic peptides derived from TAAs may provide vaccine platforms that can enhance immunogenicity and reduce toxicity such as autoimmune toxicity.
  • a TAA payload may be an oncofetal antigen that is typically only expressed at different stages during the development of the fetus and in cancerous somatic cells. Many proteins are normally expressed during fetal development but are transcriptionally repressed after birth or at early stage of infancy, therefore are not present, or are expressed in significantly lower levels in the corresponding normal adult tissue.
  • the developmental proteins are re-expressed in certain tumor cells and become oncofetal antigens.
  • the oncofetal antigens have the potential to be used as tumor markers for diagnosis, treatment monitoring, follow-up after therapy and/or ultimately as targets for specific therapy of malignancy.
  • oncofetal antigens may include, but are not limited to CEA (carcinoembryonic antigen) in colorectal carcinorma, iLRP/OFA (immature laminin receptor protein/oncofetal antigen) in renal cell carcinoma (RCC), TAG-72 (tumor associated glycoprotein-72) in prostate carcinoma, AFP (alpha-fetoprotein) in hepatocellular carcinoma (HCC), ROR1 (a receptor tyrosine kinase) in many malignant cells such as brain tumors, sperm protein 17, HMGA2 (high mobility group A2) in ovarian carcinoma, oncofetal H19, CR-1 (Cripto-1, a member of epidermal growth factor (EGF)-CFC family), trophoblast glycoprotein precursor and GPC-3 (Glypican-3, a member of heparan sulphate
  • CEA carcinoembryonic antigen
  • iLRP/OFA immature laminin receptor protein
  • T cell epitope peptides derived from oncofetal antigens may be used as payloads, such as those peptides disclosed in U.S. Pat. NOs. :
  • a TAA payload may be an oncoviral antigen that is encoded by tumorigenic transforming viruses (also called oncogenic viruses).
  • Oncogenic viruses when they infect host cells, can insert their own DNA (or RNA) into that of the host cells. When the viral DNA or RNA affects the host cell's genes, it can push the cell toward becoming cancer.
  • Oncogenic viruses include, but are not limited to, RNA viruses, such as Flaviviridae and Retroviridae, and DNA viruses, such as Hepadnaviridae, Papovaviridae, specifically
  • Papillomaviruses Adenoviridae, Herpesviridae, and Poxviridae.
  • HPVs human papilloma viruses
  • EBV Epstein-Barr virus
  • HCC hepatitis B, C and D viruses
  • HCV human immunodeficiency virus
  • KSHV Kaposi sarcoma herpes virus
  • HHV8 human herpes virus 8
  • a viral antigen can be any defined antigen of a virus that is associated with a cancer in a human.
  • a viral antigen is one that results in a CD8+ T-cell response that can be readily/easily measured.
  • the viral antigen is one to which an immune response can be induced or stimulated in a human and is universally recognized.
  • EBV antigens include, but are not limited to, Epstein-Barr nuclear antigen-1 (EBNAl), latent membrane protein 1 (LMP1), or latent membrane protein 2 (LMP2).
  • suitable HPV antigens for conjugates include, but are not limited, LI and L2 protein, and E5, E6, and E7.
  • KSHV antigens for conjugates may include but are not limited to, latency nuclear antigen (LANA) and v-cyclin.
  • suitable HIV antigens include, but are not limited to gpl60, gpl20 and gag protein. It is within the scope of the present invention that any antigenic peptides derived from oncoviral antigens may be used as active pay loads of the present conjugates.
  • a TAA payload may be an overexpressed or accumulated antigen that is expressed by both normal and neoplastic tissue, with the level of expression highly elevated in cancer tissues.
  • Numerous proteins e.g. oncogenes
  • are up-regulated in tumor tissues including but not limited to adipophilin, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH, epCAM, ephA3, EZH2, FGF5, G250, HER-2/neu, HLA- DOB, Hepsin, IDOl, IGFB3, IL13Ralpha2, Intestinal carboxyl esterase, kallikrein 4, KIF20A, lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC-1 , MUC5AC, p53, Pax5, PBF, PRAME,
  • Antigenic peptides derived from TAAs that are overexpressed in tumor tissues can be found in many references. Some examples may be U.S. Pat. NO.: 7,371,840; 7, 906, 620; U.S. patent publication No. 2010/0074925; the content of each of which is incorporated herein in their entirety.
  • a TAA payload may be a cancer-testis antigen that is expressed only by cancer cells and adult reproductive tissues such as testis and placenta.
  • a TAA in this category may include, but are not limited to antigens from BAGE family, CAGE family, HAGE family, GAGE family, MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A6 and MAGE-A13), SAGE family, XAGE family, MCAK, NA88-A (cancer/testis antigen 88), PSAD1, SSX-2, and SLLP-1.
  • NY-ESO-1 is one of the most immunogenic TAAs which expression is limited to testis in healthy subjects, but often overexpressed in various cancers such as HCC, melanoma, ovarian, and breast cancer.
  • a TAA payload may be a lineage restricted antigen that is expressed largely by a single cancer histotype.
  • a lineage restricted antigen may include, but are not limited to, Melan-A/MART- 1 , Gpl00/pmell7, Tyrosinase, TRP-1/-2, P.polypeptide, MClR in melanoma; and prostate specific antigen (PSA) in prostate cancer. Any antigenic peptides derived from these TAAs may be used as active payloads of the present conjugates.
  • a TAA payload may be a mutated antigen that is only expressed by tumor cells as a result of genetic mutations or alterations in transcription.
  • the antigen may be resulted from genetic substitution, insertion, deletion or any other genetic changes of a native cognate protein (i.e. a molecule that is expressed in normal cells).
  • a subset of these mutations can alter protein coding sequences, therefore creating novel, foreign antigens: tumor neoantigen.
  • tumor neoantigens refers to tumor antigens that are present in tumor cells but not normal cells and do not induce deletion of their cognate antigen specific T cells in thymus (i.e., central tolerance).
  • tumor neoantigens may provide a "foreign" signal, similar to pathogens, to induce an effective immune response needed for cancer immunotherapy.
  • a neoantigen may be restricted to a specific tumor.
  • a neoantigen be a peptide/protein with a missense mutation (missense neoantigen), or a new peptide with long, completely novel stretches of amino acids from novel open reading frames (neoORFs).
  • the neoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in DNA mismatch repair causing microsatellite instability), gene-fusion, read-through mutations in stop codons, or translation of improperly spliced RNA (e.g., Saeterdal et al, Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer, Proc Natl Acad Sci USA, 2001, 98: 13255-13260). Studies have showed that neoORFs generated by frameshift mutations, which are not subject to central tolerance, induce highly specific antitumor immunity, and are thus highly valuable as antigens for cancer immunotherapy.
  • CTLs cytotoxic T lymphocytes
  • these neoantigens may include mutated new peptides derived from alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK 2A, CLPP, CML-66, COA-1, connexin 37, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6- AML1 fusion protein, fibronectin, FLT3-ITD, FN1, GPNM8, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, Hsp-70-lB, MART-2, ME1, MUM-1, MUM-2, MUM- 3, Myosin class I, NFYC, neo-PAP, OGT, OS-9, p53, pml-RARalpha fusion protein, PRDX5, PTPRK,
  • Additional neoantigen peptides may include SF3B1 peptides, MYD peptides, TP53 peptides, Abl peptides, FBXW7 peptides, MAPK peptides, and GNB1 peptides disclosed in US patent publication NO.: 20110293637; the content of which in incorporated herein in its entirety.
  • Tumor associated mutations are discovered rapidly through DNA and RNA sequencing of tumor and normal tissues. Massively parallel sequencing techniques can sequence the entire genome or exome of tumor and matched normal cells to identify all of the mutations that have occurred in tumor cells. The comprehensive maps of mutated antigens in tumor genomes bring new targets for therapeutic or prophylactic vaccines (Wood LD, et al, The genomic landscapes of human breast and colorectal cancers. Science, 2007, 318: 1108- 1113; PCT patent publication NO.: WO2014168874; the content of each of which is incorporated by reference in their entirety).
  • these new neoantigens identified through large-scale sequencing and algorithm calculation may be linked to conjugates of the present invention as payloads.
  • Novel tumor antigenic peptides are identified by some studies may be used as payloads of the conjugates. See, e.g., Nishimura et al, Cancer immunotherapy using novel tumor associated antigenic peptides identified by genome-wide cDNA microarray analyses, Cancer Sci. 2015, 106(5): 505-511 ; and Linnemann et al., high-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma, Nat. Med., 2015, 21(1): 81-85; the content of each of which is incorporated by reference in their entirety.
  • Conjugates comprising tumor neoantigens may be used as ideal therapeutic and prophylactic vaccines.
  • a TAA payload may be an idiotypic antigen that is generated from highly polymorphic genes where a tumor cell expresses a specific "clonotype", i.e., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies, such as Immunoglobulin and T cell receptors (TCRs).
  • Idiotypic antigens are a class of nonpathogen-associated neoantigens.
  • the malignant B cells express rearranged and multiply mutated surface immunoglobulins (Ig).
  • Tumor specific idiotypes e.g., immunoglobulin idiotypes
  • immunoglobulin idiotypes are regarded as particularly attractive tumor-specific antigens that can be successfully targeted by immunotherapy (e.g., Alejandro et al, Idiotypes as immunogens: facing the challenge of inducing strong therapeutic immune responses against the variable region of
  • a TAA payload may be a post-translationally altered antigen due to tumor - associated alterations in glycosylation, and other posttranslational modifications. Some examples may include MUC1 in colorectal carcinoma.
  • antigenic peptides and their corresponding genes/proteins, HLA subtypes to which an antigenic peptide binds and tumors associated with them are listed in Table 1 (e.g., Vanern et al, Database of T cell defined human tumor antigens: the 2013 update, Cancer Imus. 2013, 13: 15).
  • PSMA NYARTEDFF 178-186 A24 prostate, CNS, liver
  • payloads of the present conjugates may be tumor specific antigens and /or their antigenic peptides disclosed in U.S. Pat. NOs: 8,961,985; 8,951,975; 8,933,014; 8,889,616; 8,895,514; 8,889,616; 8,871,719; 8,697,631; 8,669,230; 8,647,629; 8,653, 035; 8,569, 244; 8, 455, 615; 8,492,342; 8, 318, 677; 8, 258, 260 ; 8,212,000; 8,211,999;
  • Antigenic peptides may also include those identified by methods disclosed in, e.g., US pat. NOs.: 9,090, 322; 8, 945, 573; 8, 883,164; and US patent publication NOs. :
  • TAAs and antigenic peptides may include those discussed by, e.g., Akiyama et al, Cancer Immunol. Immunother. 2012, 61 : 2311-2319; Alisa et al, J Immunol 2008, 180: 5109-5117; Alves et al., Cancer Res 2003; 63: 8476-8480; Anderson et al., Cancer Res 2004, 64: 5456-5460; Bae et al, Br J Haematol 2012, 157: 687-701; Belle et al, Eur J Haematol 2008, 81 : 26-35; Bund et al, Exp Hematol 2007, 35: 920-930; Chen et al, Neoplasia 2008, 10: 977-986; Coleman et al., Int J Cancer 2011, 128: 2114-2124; Dong et al., Cancer Lett 2004, 211 : 219-25; Erfurt et al, Int J Cancer
  • payloads of the conjugates may be TAA or antigenic peptide analogs.
  • An antigenic peptide analog such as a neoantigen analog may be a molecule that is not identical, but retains the biological activity (e.g., immunogenicity) and/or has analogous structural features to a corresponding naturally occurring tumor specific antigen such as neoantigen.
  • TAA and antigenic peptide analogs may be substituted and/or homologous peptides related to a naturally occurring antigenic peptide, such as altered peptide ligands (Kersh and Allen, Essential flexibility in the T-cell recognition of antigen. Nature. 1996, 380: 495-498).
  • Those substitutes and homologs retain similarities to the original peptides and are recognized in a highly similar fashion (e.g., Macdonald et al, T cell allorecognition via molecular mimicry. Immunity. 2009, 31 : 897-908).
  • the peptide analogs are intended to increase characteristics of naturally occurring antigenic peptides such as resistance against peptide degradation and enhancing the activity of the native epitope to induce cytotoxic T lymphocytes.
  • TAA or antigenitc peptide analogs may be biochemically modified as necessary to provide some desired attributes such as improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified antigenic peptides to bind the desired MHC molecules and activate the appropriate T cells.
  • Such modifications may also increase the protease resistance, membrane permeability, or half-life without altering, for example, ligand binding.
  • a TAA or an antigen peptide may be subject to various modifications, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC molecule binding.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, He, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids.
  • Such modifications may be made using well known peptide synthesis procedures, as described in e.g., Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III, Pierce), 2d Ed. (1984).
  • the TAA and antigenic peptide may also be modified by extending or decreasing the amino acids of the peptide, such as by the addition or deletion of amino acids.
  • an antigenic peptide may include amino acid minics and unnatural amino acids, such as 4-chlorophenylalanine, D- or L-naphylalanine; D- or L- phenylglycine; D- or L-2-thieneylalanine; D- or L-l, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2- pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2- pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro- methyl)-phenylalanine; D-p-fluorophenylalanine; D- or L-p-biphenyl- phenyla
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides with amino acid mimetics or unnatural amino acid residues may manifest increased stability in vivo.
  • an antigenic peptide may be modified by N-terminal acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, and/or C-terminal amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for connecting to a linker within the conjugate.
  • a mixture of antigenic peptides derived from a single TAA may be used as pay loads of the present conjugates.
  • the peptide mixture may be a mixture of HLA class I specific epitopes and HLA class II specific epitopes.
  • more than one antigenic peptide may be included into a conjugate.
  • the peptides may be selected from a spectrum of different antigens that are associated with a particular cancer.
  • Multiple TAA payloads may enhance the coverage of tumor antigens from a target cancer and therefore enhance the capability of antigen presentation and infiltrate sufficient effector T cells to kill tumor cells.
  • There are several advantages using multiple antigens including i): increasing likelihood of generating a robust immune response against at least some of the antigens; and ii): decreasing the likelihood of a tumor escaping the immune response by immunoediting, because it must downregulate multiple targets.
  • two, three, four, five, six or seven antigens from a list of known HCC specific antigens: alpha-fetoprotein (AFP), glypican-3 (GPC3), NY- ESO-1, SSX-2, melanoma antigen gene-A (MAGE-A), telomerase reverse transcriptase (TERT), and hepatocellular carcinoma-associated antigen-519/targeting protein for Xklp-2 (HCA519/TPX2), may be selected as payloads of a conjugate.
  • conjugates may enhance an immune response against HCC tumor cells.
  • Conjugates comprising antigen payloads may comprise at least two or more neoantigenic peptides.
  • the composition contains at least two distinct peptides.
  • the at least two distinct peptides are derived from the same polypeptide (e.g., the same TAA).
  • distinct polypeptides is meant that the peptide vary by length, amino acid sequence or both.
  • payloads of the conjugates of the present invention may comprise between 1 to 20 antigen peptides, for example, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different antigen peptides. In other aspects, more than 20 antigen peptides may be included in the conjugates as payloads.
  • antigen payloads may be "personalized” tumor antigens from a subject who has a tumor.
  • personalized tumor antigens refers to individual patient specific neoantigens that are encoded by a collective of the individual patient's tumor-specific alternations and mutations.
  • tumor antigen payloads may be "shared” tumor antigens.
  • shared tumor antigens refers to a collective of neoantigens that are commonly presented in a specific type of tumor for example breast tumor.
  • TAA-derived CD4+ T helper cell epitopes may be induced in a conjugate along with CD8+ T-cell epitopes.
  • TAAs may be lipid molecules, polysaccharides, saccharides, nucleic acids, haptens, carbohydrate, or the combinations thereof. 2. APC activation, maturation and migration
  • APCs Antigen presenting cells
  • DCs dendritic cells
  • payloads of the present conjugates may be any active agents that can increase APCs (i.e. DCs) activity.
  • the active agents may function at any step during the process of dendritic cell maturation, migration, activation and antigen presentation, and/or cytokine production.
  • a payload may be an active agent that can promote DCs recruitment, maturation and migration along the lymphatic vessels and into the Lymph Node (LN) (e.g., tumor draining lymph node), therefore, promoting scanning a vast T cell repertoire within the LN.
  • LN Lymph Node
  • a payload may be an agent that can enhance antigen presentation of DCs, i.e. converting antigens into peptide-MHC complexes.
  • the active agent may increase antigen uptake from e.g., death cells of tumors, and efficiently extract peptides from them.
  • an active agent may be a chemokine that binds to a chemokine receptor on DCs to regulate DCs.
  • Migration of antigen loaded dendritic cells into lymphatic vessels to lymph node to encounter T cells requires chemokine stimulation and induction of the chemokine receptors (e.g., CCR7).
  • DCs express a panel of inflammatory chemokine receptors including CCRl, CCR2, CCR4, CCR5, CCR6, CCR 8, CCR9, CXCR3, CX3CR, CXCR4 and CCR7, each of which binds to one or more ligands to regulate different aspects of DC maturation, migration, and interaction with naive T cells in lymph nodes.
  • Some ligands that bind to and activate these receptors include, but are not limited to, CCL3 ( ⁇ ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL9 (MRP -2), CCL14 (HCC1), CCL16 (HCC4) which are CCRl ligands; CCL2 (MCP-1), CCL7 (MCP-3), CCL12 (MCP-5), CCL8 (MCP-2), CCL16 (HCC4) which are CCR2 ligands; CCL17 (TARC), CCL19 ( ⁇ -3 ⁇ , ELC) which are CCR4 ligands; CCL3 (MP la), CCL4 ( ⁇ ), CCL5 (RANTES), CCL8 (MCP-2), CCL11 (eotaxin), CCL14 (HCC1), CCL16 (HCC4) which are CCR5 ligands; CCL20 (MIP-3a), a ligand of CCR6; CCL1 (TCA3), a
  • the chemokine receptor CCR7 on DCs when binding to its ligand CCL19 and CCL21 can regulate the migratory speed of DCs, directing DCs to secondary lymphoid nodes and to elicit an adaptive immune response.
  • the chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immnuno., 2007, 174(7):4070-80; and Verdijk et al, Maximizing dendritic cell migration in cancer immunotherapy. 2008, Expert Opin Biol Ther., 8(7): 865-874).
  • a payload may be a cytokine that can stimulate/regulate the expression both MHC/HLA class I and class II molecules on APCs (i.e. DCs).
  • Interferon- ⁇ IFN- ⁇
  • Interferons also enhance the antigen presenting function of MHC/HLA class I molecules by inducing the expression of key components of the intracellular machinery that enables peptides to be loaded onto the MHC molecules.
  • Payloads may also be other agents that can stimulate and induce antigen presenting function of other cells for example, ⁇ T cells.
  • ⁇ T cells may include isopentenyl pyrophosphate (IPP) and others disclosed by Brandes et al (US Pat. No. : 8, 153, 426, which is incorporated herein by reference in its entirety).
  • antigen presentation is reduced or impaired due to impairment of one or more components of MHC class I/II antigen presenting pathway.
  • mutations which cause a reduced expression of a component e.g., reduced expression of MHC class I gene due to changes in methylation or chromatin structure, or cause a mutated component that has reduced or no function.
  • Impairments in these components typically affect processing (e.g., proteolysis) of proteins to form peptide epitopes, or transporting peptide to the endoplasmic reticulum, or formation or transport of peptide/MHC molecule (pMHC) complex to the cell surface.
  • components may be MHC class I alpha chain polypeptide, beta2m macroglobulin and TAP.
  • the payload of the conjugate may be a MHC/HLA molecule or a variant thereof that contains sequences to match any known TAA or peptide epitope.
  • Conjugates comprising such molecules may mimic DC derived function to directly activate CD8+ and CD4+ T cells inducing a strong immunogenic response against tumor.
  • the antigen presenting molecules may be MHC/HLA class I or class II molecules.
  • MHC/HLA class I molecules are cell surface glycoproteins and are heterodimeric and composed of a polymorphic, MHC-encoded, approximately 45 kD a chain, which is non- covalently associated with an approximately 12 kD ⁇ -2 microglobulin ( ⁇ -2 ⁇ ).
  • the extracellular portion of the MHC Class I a chain is divided into three domains, a-1, a-2, and a-3, each approximately 90 amino acids long and encoded on separate exons.
  • the a-3 domain and ⁇ -2 ⁇ are relatively conserved and show amino-acid sequence homology to
  • the polymorphic a-1 and a-2 domains show no significant sequence homology to immunoglobulin constant or variable region.
  • the polymorphic a-1 (approximately 90 amino acids) and a-2 (approximately 92 amino acids) domains are responsible to antigen recognition.
  • the a-2 domain is attached to the less- polymorphic, membrane-proximal a-3 (approximately 92 amino acids) domain which is followed by a conserved transmembrane (25 amino acids) and an intra-cytoplasmic
  • the classical class I gene family includes the highly polymorphic human class I molecules HLA-A, HLA-B, and HLA-C.
  • HLA-A, -B, and -C genes encode molecules that bind antigenic peptides, and present the peptides to CD8 + T cells, thereby initiating a cytotoxic T cell (CTL) response during infection.
  • CTL cytotoxic T cell
  • Extensive allelic polymorphisms are observed in the HLA-A, B and C genes, concentrated primarily among nucleotides that encode residues within the peptide binding grooves of the HLA class I molecules, which determine specificity for the associated peptide ligands.
  • payloads may be a polypeptide encoded by any of the known HLA genetic loci, as well as polypeptides encoded by genetic loci not yet discovered so long as these can present antigen to a T cell in a manner effective to activate the T cell receptor.
  • HLA-A A*01, A*02, A*03, A*l l, A*23, A*24, A*25, A*26, A*28, A*29, A*30, A*31, A*32, A*33, A*34, A*36, A*43, A*66, A*68, A*74 and A*80;
  • HLA-B B*07, B*08, B* 13, B* 14, B* 15, B*18, B*27, B*35, B*37, B*38, B*39, B*40, B*41, B*42, B*44, B*45, B*46, B*47, B*48, B*49, B*50, B*51, B*52, B*53, B*54, B*55, B*56, B*57, B*58, B*59, B*67, B*73, B*78, B*81, B*82 and B*83; and for HLA-A: A*01, A*02, A*
  • polypeptides of HLA class II a and ⁇ chain proteins may include polypeptides from genetic loci for HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA and HLA-DPB.
  • the MHC/HLA polypeptides selected for inclusion in the present conjugates may also include polypeptide variants such as a modified polypeptide.
  • conjugates comprising HLA-A, HLA-B, HLA-C, TAP and beta2m polypeptides may be delivered to tumor cells to restore antigen presentation in tumor cells, therefore activate and expand tumor specific cytotoxic T lymphocytes (CTL) to kill tumor cells.
  • CTL cytotoxic T lymphocytes
  • HLA-A, HLA-B and HLA-C, TAP and beta2m payloads of the conjugates may be connected to a targeting moiety through the linker.
  • Such conjugates in some aspects, may be fused or co-conjugated with one or more TAAs or peptide epitopes.
  • the peptide- MHC molecule (pMHC) complexes may be delivered to a subject directly targeted to tumor cells.
  • CD40 Activated antigen-presenting B cells have been shown to efficiently induce both CD4 + and CD8 + T cells responses in vitro and in vivo.
  • B cell-based vaccines as an alternative to DC-based vaccines for cancer immunotherapy (von Bergwelt-Baildon et al., Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application, Blood, 2012, 99:3319-3325).
  • the conjugate of the present invention may comprise an active agent that can activate B cell antigen
  • effector T cells e.g. CD4+ T cells and CD8+ T cells
  • a payload may an agent that can active effector T cells, or assist T cells in killing tumor cells, or increase the specificity of effector T cells to specific tumor cells.
  • the active agent may be an agent that can enhance TAA processing and presentations such as other signals that are provided to T cells by natural antigen presenting cells (APCs). T cell immune responses are mediated by the signals received from APCs.
  • TCR T cell receptor
  • pMHC peptide/major histocompatibility complex
  • active agents of the present conjugates may be one or more co-stimulatory agents.
  • co-stimulatory agents may impact expansion, survival, effector function, and memory of stimulated T cells
  • the co-stimulatory agents may include but are not limited to antigens, polyclonal T cell receptor activators, co-stimulatory and targeting molecules, and cytokines, which allow for control over the signals provided to T cells by natural APCs. These fully activated signals can be transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells and induction of cytotoxicity or cytokine secretion.
  • the active agent may be a polyclonal T cell receptor activator.
  • a polyclonal TCR activator can activate T cells in the absence of specific antigens.
  • Suitable polyclonal T cell activators include the mitogenic lectins concanavalin-A (ConA), phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and antibodies that crosslink the T cell receptor/CD3 complex.
  • Exemplary antibodies that crosslink the T cell receptor include the HIT3a, UCHT1 and OKT3 monoclonal antibodies.
  • the active agent may be a co-stimulatory molecule, or any compound that has similar function.
  • Activation and proliferation of T cells are also regulated by both positive and negative signals from costimulatory molecules.
  • One extensively characterized T cell costimulatory pathway is B7-CD28, in which CD80 (B7-1) and CD86 (B7-2) on APCs can interact with stimulatory CD28 receptor and the inhibitory CTLA-4 (CD152) receptor on T cells, respectively.
  • CD28 ligation increases antigen-specific proliferation of T cells, enhances production of cytokines, stimulates differentiation and effector function, and promotes survival of T cells.
  • a conjugate of the present invention may comprise at least one costimulatory molecule or agent that can stimulate those co-stimulatory effects, as an active agent to be connected to the targeting moiety through the linker.
  • the term "co-stimulatory molecule”, in accordance with its meaning in immune T cell activation refers to a group of immune cell surface receptor/ligands which engage between T cells and APCs and generate a stimulatory signal in T cells which combines with the stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs.
  • TCR T cell receptor
  • pMHC antigen/MHC complex
  • co-stimulatory molecules also referred to as "co-stimulators” include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL receptor (CD137), 4-1BB ligand (CD137-L), OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD2, CD5, CD9, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, glucocorticoid- induced tumor necrosis factor receptor ligand (GITR-L), an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
  • co-stimulators include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL receptor (CD137),
  • co- stimulatory molecules that can be used include antibodies that specifically bind with a co- stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
  • suitable costimulatory molecules include, but are not limited to, costimulatory variants and fragments of the natural ligands described above.
  • a variant may be a soluble form of a co-stimulatory molecule.
  • the soluble form of a co-stimulatory molecule is a fragment of a full length costimulatory molecule only containing one or more extracellular domains of the co-stimulatory molecule (e.g., U. S. Pat No. : 8, 268,788).
  • the soluble form of a co-stimulatory molecule derived from an APC retains the ability of the native co-stimulatory molecule to bind to its cognate receptor/ligand on T cells and stimulate T cell activation.
  • a non-limiting example is a soluble form of CD137-L.
  • the active agent of the conjugate may be a T cell adhesion molecule that can increase the binding association between the antigen-loaded/activated APCs and T cells.
  • Suitable adhesion molecules include, but are not limited to, CDl la (LFA- 1), CD 11c, CD49d/29(VLA-4), CD50 (ICAM-2), CD54 (ICAM-1), CD58 (LFA-3) CD 102 (ICAM-3) and CD106 (VCAM), and antibodies to their ligands.
  • Other suitable adhesion molecules include antibodies to selectins L, E, and P.
  • the active agent of the conjugate may be a cytokine or other immunoregulatory agent.
  • Cytokines may be secreted by activated APCs after T cell encounters and impact expansion, survival, effector function, and memory of stimulated T cells.
  • at least one cytokine may be connected to the targeting moiety through the linker.
  • Suitable cytokines include, but are not limited to, hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules and chemokines.
  • cytokines include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFa), tumor necrosis factor beta ( ⁇ ), macrophage colony stimulating factor (M-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), interferon alpha (IFNa), interferon beta ( ⁇ ), interferon gamma (IFNy), and interferon-gamma inducing factor (IGIF), and variants and fragments thereof.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TNFa tumor necrosis factor alpha
  • ⁇ tumor necrosis factor beta
  • M-CSF macrophage colony stimulating factor
  • TAAs and/or antigenic peptides derived from TAAs, costimulatory factors, T cell adhesion molecules and cytokines secreted by activated APCs may be connected to the targeting moiety through the linker in one conjugate.
  • conjugates comprising each individual agent may be packaged into one particle or a formulation of the present invention.
  • a payload may be a T cell receptor (TCR) or a TCR analog (e.g., engineered CAR) having antigenic specificity for a TAA, e.g., any antigen peptide as discussed above.
  • TCR T cell receptor
  • a TCR analog e.g., engineered CAR
  • Mature T cells express a unique ⁇ TCR that can bind to peptides presented by MHC molecules. Unlike antibodies, TCRs generally have low affinity for ligands, facilitating a rapid scanning of antigen peptide-MHC complexes.
  • CDR3 loops of a TCR primarily engage the binding with antigen peptide presented in the MHC groove, while CDR1 and CDR2 loops can contact with the tops of the MHC helices (Garcia and Adams, How the T cell receptor sees antigen-a structural view. Cell. 2005, 122: 333-336; Rudolph et al, How TCRs bind MHCs, peptides, and coreceptors. Annual Review of
  • Tumor specific TCRs may be obtained from spontaneously occurring tumor-specific T cells in patients, such as the melanocyte differentiation antigens MART-1 and gplOO, as well as the MAGE antigens and NY-ESO-1, with expression in a broader range of cancers. TCRs may also be isolated from viral infected cells in some viral-associated malignancies. Additionally, TCRs specific to a TAA may also be identified by, for example, allogeneic TCR and transgenic mice expressing human a HLA molecule.
  • recombinant technology can be used to generate TCRs on phage display libraries, which can be used to identify novel high affinity tumor-specific TCRs (Zhao et al, High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines. J Immunol. 2007, 179:5845-5854). Isolated TCRs may be used as active agents of the conjugates of the present invention.
  • a TCR active agent of the conjugate of the present invention may be a CDR3 region peptide of TCR against a specific TAAs such as WT-1 as disclosed in US patent publication NO. 2014/0315735; the content of which is herein incorporated by reference in its entirety.
  • the TCR may be ⁇ T-cell receptors consisting of a ⁇ chain and a ⁇ chain polypeptide
  • ⁇ T-cell receptors may be specialized to bind certain kinds of ligands, including heat-shock proteins and nonpeptide ligands such as mycobacterial lipid antigens. It seems likely that ⁇ T-cell receptors are not restricted by the 'classical' MHC class I and class II molecules. They may bind the free antigen, much as immunoglobulins do, and/or they may bind to peptides or other antigens presented by non-classical MHC-like molecules. These are proteins that resemble MHC class I molecules but are relatively nonpolymorphic.
  • a TCR analog may be a chimeric antigen receptor (CAR) that can recognize a specific cell surface tumor antigen independent of MHC/HLA molecules and employs one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production.
  • An engineered chimeric antigen receptor (CAR) may be composed of an antibody-derived targeting domain (i.e., an extracellular domain derived from tumor-specific antibody) fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR.
  • the targeting domain of a CAR may be derived from any antibody that specifically recognizes a tumor specific antigen.
  • a single-chain variable fragment (ScFv) of antibodies are used in the extracellular domain of CARs, which are joined through hinge and transmembrane regions to intracellular signaling domains.
  • Tumor-specific antibodies may be generated through immunization of mice. Recombinant techniques can be used to humanize antibodies, or mice expressing human immunoglobulin genes can be used to generate fully human antibodies.
  • T cell activation is a complex process involving several signals including a primary initiating signal and secondary costimulatory signals.
  • Inclusion of such signals in CARs can enable responses against cancer cells.
  • inclusion of a primary signaling molecule CDS- ⁇ in CARs can induce T cell activation.
  • Inclusion of the cytoplasmic domain of CD28, CD134 or 4-1BB (CD137) in CARs can lead to increased cytokine production in response to a TAA (e.g., Carpenito et al, Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and 4-1BB (CD137) domains. Proc Natl Acad Sci USA. 2009, 106:3360-3365).
  • CARs specific for a wide range of TAAs have been developed, for example, CD 19 specific CAR for leukemia (Kochenderfer et al, adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD 19 can eradicate lymphoma and normal B cells. Blood, 2010, 116: 3875-3886), Chmielewski et al., T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology. 2012, 143: 1095-1107; Westwood et al. Adoptive transfer of T cells modified with a humanized chimeric receptor gene inhibits growth of Lewis-Y-expressing tumors in mice. Proc Natl Acad Sci USA. 2005, 102: 19051- 19056).
  • the active agent of the conjugate may be co-receptors of TCRs such as CD4 and CD8.
  • the payload may be a full length of co-receptors CD4 and CD8, or a domain thereof that can bind to a MHC/HLA molecule.
  • the payload may be a CD4 immunoglobulin-like domain that can bind to an invariant site of the MHC class II molecule, such as the ⁇ 2 domain.
  • the payload may be a CD8 domain that can bind to an invariant site of the MHC class I molecule, such as the a3 domain.
  • CD4 and CD8 co-receptors that bind to MHC class II and I molecules respectively, can markedly increase the sensitivity of a T cell to antigen presented by MHC molecules on APCs.
  • Conjugates comprising TCRs, CARs or co-receptors, or variants thereof may be used to engineered T cells for adoptive immunotherapy.
  • adoptive T cell immunotherapy A detailed discussion of adoptive T cell immunotherapy is described in the following sections.
  • the active agent of the conjugate is a CD3-binding agent, such as a peptide or derivative that binds to CD3, a CD3 antibody or a CD3-binding fragment thereof.
  • Activation of cytotoxic T cell may occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by the conjugates of the present invention.
  • CD3 (cluster of differentiation 3) complex, or CD3 antigen is a T cell co-receoptor that helps to activate T cells.
  • CD3 complex may comprise several chians: CD3D (CD3 delta chain), CD3G (CD3 gamma chain), CD3E (CD3 epsilon chain) and/or CD247 (CD3 zeta chain).
  • CD3D CD3 delta chain
  • CD3G CD3 gamma chain
  • CD3E CD3 epsilon chain
  • CD247 CD3 zeta chain
  • the CD3-binding agent, CD3 antibody or the CD3-binding fragment may bind to any epitope on any of the chains.
  • CD3 antigens are cell-surface proteins and are bound to the membrances of all mature T cells.
  • Conjugates of the present invention comprising CD3 binding agents may bind to and activate T cells in the absence of independent TCR/MHC binding. The activated T cell can then exert a cytotoxic effect on tumor cells.
  • CD3 antigents do not internalize upon binding of the conjugates.
  • the CD3 binding agent may be a Fab fragment of a CD3 antibody, a single CDR CD3 antibody, a single chain variable fragment (scFv) of a CD3 antibody, a single-chain antibody mimic that is much smaller than an antibody such as nanofitin® (Affilogic).
  • CD3 antibodies or fragments thereof include, a humanized CD3-specific scFv disclosed by Liddy et al. (Nature Medicine, vol. l8(6):980 (2012)), a single-chain anti-CD3 antibody derived from UCHT1 disclosed by Kuo et al.
  • an anti-CD3 scFv comprising an amino acid sequence of SEQ ID No.2 in CA2561826 to Wang et al., an anti-CD3 portion of an anti-CD3&anti-EpCAM bispecific antibody (SEQ ID No. l) disclosed in WO2005061547 to Baeuerle et al, a reshaped Fab antibody against human CD3, a reshaped single-domain antibody against human CD3 or a reshaped scFv against human CD3 disclosed in US20050175606 to Huang et al, anti-CD3 VH disclosed in
  • the active agent of the conjugate activates other effector cells, such as natural killer cells.
  • the active agent of the conjguate is a CD 16 antibody or a CD16-binding fragment thereof.
  • CD 16 is an Fc receptor found on the surface of natural killer cells.
  • Conjugates of the present invention binds to CD16 on natural killer cells and activate natural killer cells.
  • Non-limiting examples of CD 16 antibodies or CD16-binding fragment thereof include monoclonal antibody of the IgGl class against human CD 16 antigen disclosed in US5643759 to Pfreundschuh, FV
  • the active agent of the conjuate binds to a universal CAR T cell and activates the CAR T cell.
  • the binding between the active agent and the CAR T cell may occur only in the tumor microenvironment, or is activated by light, heat, radiation, or chemical agents such as but not limited to tetrac cline,
  • the binding site on the CAR T cell, or the active agent may comprise a masking moiety described herein.
  • the binding of the active agent to the CAR T cell may be inhibited or hindered by the masking moiety.
  • the binding may be sterically hindered by the presence of the masking moiety or may be inhibited by the charge of the masking moiety.
  • Cleavage of the masking moiety, a conformation change, or a chemical transformation may unmask/activate the binding site on the CAR T cells or the active agent.
  • the masking/unmasking process may be reversible or irreversible.
  • CAR T cells may be constructed by fusing an anti- fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137.
  • the active agent may comprise fluorescein. Therefore, the active agent binds to the CAR T cells and activates T cell cytotoxcity.
  • a payload of a conjugate of the present invention may be an immunoregulatory molecule.
  • Conjugates may comprise more than one immunoregulatory molecules as payloads, e.g., two, three, four, five, six, seven or more immunoregulatory molecules.
  • immunoregulatory cytokines include, but are not limited to, interferons (e.g., IFNa, ⁇ and IFNy), interleukins (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-20), tumor necrosis factors (e.g., TNFa and ⁇ ), erythropoietin (EPO), FLT-3 ligand, glplO, TCA-3, MCP-1, MIF, ⁇ - ⁇ , ⁇ - ⁇ , Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G- CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments thereof.
  • the most preferred immunomodulatory cytokine is GM-CSF, such as human GM-CSF, including
  • immunomodulatory chemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor
  • chemokines include, but are not limited to, MIP-3a (Lax), ⁇ -3 ⁇ , Hcc-1, MPIF-1, MPIF-2, MCP-2, MCP-3, MCP-4, MCP-5, Eotaxin, Tare, Elc, 1309, IL-8, GCP-2 Groa., Gro- ⁇ ., Gro- ⁇ , Nap-2, Ena-78, Ip-10, MIG, I-Tac, SDF-1, and BCA-1 (Blc), as well as functional fragments thereof.
  • an immunoregulatory payload may be a T cell growth factor, derivative thereof, or any agent that can stimulate T cell proliferation and/or enhance T cell survival during an immune response, resulting in a more effective immune response and increased memory T cell function.
  • T cell growth factors may include, but are not limited to, interleukin (IL)-2, IL-7, IL-IL-9, IL-12, IL-14, IL-15, IL-16, IL-21 and IL-23.
  • the active agent may be IL-12 alone, or 2 interleukins in different combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.
  • an immunoregulatory payload may be a cytokines that can provide a stimulating environment for T cells differentiation.
  • cytokines that can provide a stimulating environment for T cells differentiation.
  • Naive CD4 + T cells have the capacity to differentiate into either polarized Thl, Th2 or ThO cells with the capacity to produce type 1 (IFN- ⁇ ), type 2 (IL-4) or type 0 (IFN-y+IL-4) cytokines, respectively.
  • a payload of a conjugate of the present invention may be any other immunomodulator that can modulate the activity of the immune system.
  • immunomodulator can be a cytokine, a chemokine or an adjuvant, for example, obtained from any suitable source, such as a mammal, e.g., a human.
  • the cytokine payload may be a full length of a cytokine or functional variants thereof.
  • the term "functional variant” as used herein is synonymous with "biologically equivalent variant, "biologically equivalent derivative,” or “biologically equivalent analog”.
  • a function variant may be a functional portion, fusion, or variant of a cytokine, e.g., is capable of engaging respective receptors and initiating signal transduction. Examples of function variants include cytokines lacking their signal peptides, conservative amino acid substitutions, or amino acid substitution at non-essential regions.
  • cytokine payloads may be a recombinant interferon (rSIFN-co) with changed spatial configuration disclosed by Wei (PCT patent publication No. WO2014/106459, the content of which is incorporated herein by reference it its entirety).
  • rSIFN-co recombinant interferon
  • a payload may be an antibody, a fragment of an antibody or a derivative thereof.
  • Antibodies may be immuno-specific for a tumor cell antigen or against immuno-modulatory factors.
  • An antibody that can recognize a TAA and/or a TAA antigenic peptide may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be generated by standard hybridoma techniques, phase display and recombinant techniques.
  • antibodies may recognize tumor antigens that are overexpressed in tumor cells, or tumor antigens associated with Leukaemias and lymphomas such as cell
  • CD antigens e.g. CD19, CD20, CD21, CD25 and CD37 in non-hodgkin lymphoma, CD33 in acute myeloid leukemia; CD5 in T cell leukemia, or glycoproteins on the cell surface.
  • antibodies may recognize non protein antigens such as glycolipids, e.g., ganglioside, and carbohydrates that are associated with tumors.
  • antibodies may recognize any one of TAAs as discussed hereinabove.
  • antibodies that can recognize a specific antigen epitope may include, without limitation, anti-HER2, anti-EGFR as disclosed in US Pat. No.: 9,023,362 and 8,722, 362; anti-FcyRIIB as disclosed in US Pat. No. : 8, 784,808; and antibodies against PSCA (prostate stem cell antigen) as disclosed in US Pat. No. : 8, 404, 817;
  • a payload may be an agonist antibody that can manipulate a process of a cancer specific immune response.
  • an agonist antibody may be an antibody specific to 4-lBB (CD137) (e.g., PCT patent publication NO.
  • the active agent of the conjugate may be an agonist antibody that specifically binds to an costimulatory molecule selected from CD28, B7-1 (CD80), B7-2 (CD86), 4-lBB (CD137), 4-lBB ligand (CD137-L), OX40, OX40L, inducible co-stimulatory ligand (ICOS-L), ICOS, intercellular adhesion molecule (ICAM), CD30, CD30L, CD40, CD27, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, GITR, GITR-L, TLR agonist, B7-H3, B7-H3 ligand, CD226, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2D, and DNAM-1.
  • an costimulatory molecule selected from CD28, B7-1 (CD80), B7-2 (CD86), 4-lBB (CD137
  • the active agent of the conjugate may be an antagonist antibody that specifically binds to a coinhibitory molecule selected from CTLA-4, PD-1, PD-L1, PD- L2, TIM-3, LAG-3, BTLA, CD160, C200R, TIGIT, KLRG-1, KIR, 2B4/CD244, VISTA and Ara2R.
  • a coinhibitory molecule selected from CTLA-4, PD-1, PD-L1, PD- L2, TIM-3, LAG-3, BTLA, CD160, C200R, TIGIT, KLRG-1, KIR, 2B4/CD244, VISTA and Ara2R.
  • an antibody payload may be a bispecific antibody (bsAb) or multiple specific antibody (msAb) (Wei die et al, Tumor- Antigen-Binding Bispecific Antibodies for Cancer Treatment, Seminars in Oncology, 2014, 41(5): 653-660).
  • bsAb bispecific antibody
  • msAb multiple specific antibody
  • the term "bispecific antibody” refers to an antibody construct that is capable of redirecting immune effector cells to the tumor microenvironment. Clinical studies of various bsAb constructs have shown spectacular results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses.
  • Some examples of bispecific antibodies include bispecific antibody against TIM-3 and PD-1 in WO201159877 to Kuchroo et al, the content of which is incorporated by reference in its entirety.
  • payloads may be cell surface antigens or fragments thereof.
  • the cell surface antigens may be tumor antigents, which are present by MHC I or MHC II molecules on the surface of tumor cells.
  • Tumor antigens may be tumor specific antigens (TSA), which are present only on tumor cells and not on any other cells, or tumor associated antigens (TAA), whch are present on some tumor cells and also some normal cells.
  • TSA tumor specific antigens
  • TAA tumor associated antigens
  • Tumor antigens may be cancer testis antigens (CTAs), melanocyte
  • Neoantigens refers to tumor-specific antigens derived from mutated proteins that are present only in the tumor. Neoantigens may be identified with any suitable method known in the art, such as reverse immunology comprising the steps of mutanome screening of a subject using massive parallel sequencing (MPS), computational eptitope prediction, and experimental validation of cancer neoantigens disclosed by Yoshimura et al. in J. of Clinical & Cellular Immunology, vol.6:2 (2015), the contents of which are incorporated herein by reference in their entirety. .
  • MPS massive parallel sequencing
  • the cell surface antigens may be recognized by the immune system of a subject.
  • Conjugtes of the present invention comprising such cell surface antigens and targeting moieties attach to a group of target cells in the subject, turning the cells into antigen- presenting cells (APCs) and allowing the cells to be recognized by the immune system of the subjct.
  • APCs antigen- presenting cells
  • the attachement of the conjugates of the present invention to the target cells may be in vivo or ex vivo.
  • the receptors on the target cells that bind to the targeting moieties of the conjugates do not internalize after the attachment.
  • cytotoxic agents may be used as payloads (referring to US6572856) (induce innate immune response to destroy cancer cells).
  • cytotoxic agents may include, but are not limited to maytansinoids, auristatins, calicheamicins, CC-1065, duocarmycins, anthracyclines, and doxorubicin derivatives.
  • cytotoxic agents may be cytotoxic protein including diphtheria toxin, Pseudomonas exotoxin, or cytotoxic portions or variants thereof [00167]
  • the active agent of the conjugate of the present invention may be a complement component (e.g., 21 plasma protein C3b)
  • the active agent of the conjugate of the present invention may further include an immunomodulatory adjuvant.
  • the immunomodulatory adjuvants are molecules that can increase the immunogenicity of a TAA or conquer the immune tolerance in the tumor microenvironment. (Sun and Liu, Listeriolysin O as a strong immunogenic molecule for the development of new anti -tumor vaccines. Hum Vaccin Immunother, 2013, 9(5): 1058-1068).
  • a payload of a conjugate may be a TLR (toll like receptor) agonist.
  • TLR agonist refers to a compound that acts as an agonist of a TLR. TLR agonists can trigger broad inflammatory responses that elicit rapid innate immune response and promote the activation of the adaptive immune response.
  • TLR agonists include, but are not limited to, polyinosinic acid (poly I:C), an agonist for TLR3; Cytosine-phosphorothioate-guanine (CpG), an agonist for TLR9; imiquimod, a TLR-7 agonist; resiquimod, a TLR-7/8 agonist; loxoribine, a TLR-7/8 agonist; sialyl-Tn (STn), a carbohydrate associated with the MUCI mucin on a number of human cancer cells and a TLR4 agonist; monophosphoryl lipid A (MPL), a TLR-4 agonist; FSL-1 , a TLR-2 agonist; CFA, a TLR2 agonist and Pam3Cys, a TLR-1/2 agonist.
  • poly I:C polyinosinic acid
  • CpG Cytosine-phosphorothioate-guanine
  • STn sialyl-Tn
  • STn sialyl
  • a TLR agonist may be a TLR1 agonist, a TLR2 agonist, a TLR 3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR 10 agonist.
  • a TLR agonist may be an agonist disclosed in U. S. Pat. No. : 7,993,659, which is incorporated herein by reference in its entirety.
  • a payload of the conjugate of the present invention may be mifamurtide.
  • Mifamurtide muramyl tripeptide phophatidylethanolamine (MTP-PE)
  • MTP-PE muramyl tripeptide phophatidylethanolamine
  • MDP muramyl dipeptide
  • Mifamurtide has a longer half-life than MDP, but has similar pharmacological behaviors.
  • the intracellular partem recognition molecule NOD2 detects mifamurtide and enhances NF- ⁇ signaling.
  • conjugates of the present inventiom comprising mifamurtide can be recoganized by NOD2 and can stimulate the production of IL- ⁇ , IL-6 and TNF-a via the activation of NF- ⁇ signaling in money tes and macrophages.
  • the conjugates contain one or more linkers attaching the active agents and targeting moieties.
  • the linker, Y is bound to one or more active agents and a targeting ligand to form a conjugate, wherein the conjugate releases at least one active agent upon delivery to a target cell.
  • the linker can be a Ci-Cio straight chain alkyl, Ci-Cio straight chain O-alkyl, Ci-Cio straight chain substituted alkyl, Ci-Cio straight chain substituted O-alkyl, C4-C13 branched chain alkyl, C4-C13 branched chain O-alkyl, C2-C12 straight chain alkenyl, C2-C12 straight chain O-alkenyl, C3-C12 straight chain substituted alkenyl, C3-C12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polygly colic acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl, heterocyclic, succinic ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bypiridine, indole derivative acting as a cross linker, chelator
  • the linker can be a C3 straight chain alkyl or a ketone.
  • the alkyl chain of the linker can be substituted with one or more substituents or heteroatoms.
  • the linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or digly colic acid.
  • the linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid.
  • the linker may be cleavable and is cleaved to release the active agent.
  • the linker may be cleaved by an enzyme.
  • the linker may be a polypeptide moiety, e.g. AA in WO2010093395 to Govindan, the content of which is incorporated herein by reference in its entirety; that is cleavable by intracellular peptidase.
  • Govindan teaches AA in the linker may be a di, tri, or tetrapeptide such as Ala-Leu, Leu- Ala-Leu, and Ala-Leu- Ala-Leu.
  • the cleavable linker may be a branched peptide.
  • the branched peptide linker may comprise two or more amino acid moieties that provide an enzyme cleavage site. Any branched peptide linker disclosed in WO 1998019705 to Dubowchik, the content of which is incorporated herein by reference in its entirety, may be used as a linker in the conjugate of the present invention.
  • the linker may comprise a lysosomally cleavable polypeptide disclosed in US 8877901 to Govindan et al, the content of which is incorporated herein by reference in its entirety.
  • the linker may comprise a protein peptide sequence which is selectively enzymatically cleavable by tumor associated proteases, such as any Y and Z structures disclosed in US 6214345 to Firestone et al., the content of which is incorporated herein by reference in its entirety.
  • the linker may be cleavable by lysozyme.
  • the cleaving of the linker is non-enzymatic. Any linker disclosed in US 20110053848 to Cleemann et al, the contents of which are incorporated herein by reference in their entirety, may be used.
  • the linker may be a non- biologically active linker represented by formula (I).
  • the linker may be a beta-glucuronide linker disclosed in US 20140031535 to Jeffrey, the contents of which are incorporated herein by reference in their entirety.
  • the linker may be a self-stabilizing linker such as a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring, disclosed in US20130309256 to Lyon et al, the contents of which are incorporated herein by reference in their entirety.
  • the linker may be a human serum albumin (HAS) linker disclosed in US 20120003221 to McDonagh et al, the contents of which are incorporated herein by reference in their entirety.
  • HAS human serum albumin
  • the linker may comprise a fullerene, e.g., Ceo, as disclosed in US 20040241173 to Wilson et al., the contents of which are incorporated herein by reference in their entirety.
  • the linker may be a recombinant albumin fused with poly cysteine peptide as disclosed in US 8541378 to Ahn et al, the contents of which are incorporated herein by reference in their entirety.
  • the linker comprises a heterocycle ring.
  • the linker may be any heterocyclic 1,3-substituted five- or six-member ring, such as thiazolidine, disclosed in US 20130309257 to Giulio, the content of which is incorporated herein by reference in its entirety.
  • the linker may be used with compositions of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.
  • the linker may be a hydrophilic linker as disclosed by Zhao et al. in PCX patent publication NO., WO2014/08025.1 ; the content of which is incorporated by reference in its entirety.
  • the hydrophilic linkers may contain phosphinate, sulfonyl, and/or sulfoxide groups to link active agents (payloads) to a cell-targeting moiety.
  • the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization.
  • linkers A variety of linkers that can be used with the present compositions and methods are described in WO 2004/010957,
  • the linker of the conjugate may be optional.
  • the active agent and the targeting moiety of the conjugated are directly connected to each other.
  • a conjugate can contain one or more targeting moieties or targeting ligands.
  • the conjugate can include an active agent with multiple targeting moieties each attached via a different linker.
  • the conjugate can have the structure X-Y-Z-Y-X where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is the active agent (payload).
  • Targeting ligands or moieties can be polypeptides (e.g., antibodies), peptides, antibody mimetics, nucleic acids (e.g., aptamers), glycoproteins, small molecules, carbohydrates, lipids, nanoparticles.
  • polypeptides e.g., antibodies
  • peptides e.g., antibodies
  • antibody mimetics e.g., antibodies
  • nucleic acids e.g., aptamers
  • glycoproteins e.g., small molecules, carbohydrates, lipids, nanoparticles.
  • a targeting moiety may particularly target a conjugate of the present invention to an immune cell, a tumor cell or a location where an anti-cancer immune response occurs.
  • the targeting moiety does not substantially interfere with efficacy of the therapeutic agent in vivo.
  • the targeting moiety itself can be an active agent.
  • the targeting moiety may contain adjuvant activity, in addition to targeted binding to a cell of interest.
  • the targeting moiety, X may be a peptide such as a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a peptide such as a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide epitope (e.g., an amino acid sequence motif) that can specifically bind to a TAA peptide
  • MHC/HLA protein HLA class I or class II
  • Peptide epitopes may be any one discussed above as payloads of the conjugates.
  • a conjugate may contain two or more the same or different antigen epitopes that are connected through a linker; the antigen epitopes will serve as active agents and targeting moieties.
  • Peptide antigens can be attached to MHC class I/II molecules by affinity binding within the cytoplasm before they are presented on the cell surface.
  • the affinity of an individual peptide antigen is directly linked to its amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence.
  • Such defined amino acid motifs may be used as targeting moieties.
  • the targeting moiety, X may be other peptides such as somatostatin, octeotide, LHRH (luteinizing hormone releasing hormone), epidermal growth factor receptor (EGFR) binding peptide, aptide or bipodal peptide, RGD-containing peptides, a protein scaffold such as a fibronectin domain, a single domain antibody, a stable scFv, or other homing peptides.
  • somatostatin such as somatostatin, octeotide, LHRH (luteinizing hormone releasing hormone), epidermal growth factor receptor (EGFR) binding peptide, aptide or bipodal peptide, RGD-containing peptides, a protein scaffold such as a fibronectin domain, a single domain antibody, a stable scFv, or other homing peptides.
  • a protein or peptide based targeting moiety may be a protein such as thrombospondin, tumor necrosis factors (TNF), annexin V, an interferon, angiostatin, endostatin, cytokine, transferrin, GM-CSF (granulocyte-macrophage colony- stimulating factor), or growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), (platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).
  • TNF tumor necrosis factors
  • annexin V an interferon
  • angiostatin angiostatin
  • endostatin endostatin
  • cytokine transferrin
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), (platelet-derived growth factor (
  • the targeting moiety is an antibody, an antibody fragment, RGD peptide, folic acid or prostate specific membrane antigen (PSMA).
  • the protein scaffold may be an antibody-derived protein scaffold.
  • Non-limiting examples include single domain antibody (dAbs), nanobody, single-chain variable fragment (scFv), antigen-binding fragment (Fab), Avibody, minibody, CH2D domain, Fcab, and bispecific T-cell engager (BiTE) molecules.
  • scFv is a stable scFv, wherein the scFv has hyperstable properties.
  • the nanobody may be derived from the single variable domain (VHH) of camelidae antibody.
  • the targeting moiety is a tumor cell binding moiety.
  • it may bind to a somatostatin receptor (SSTR) such as SSTR2 on tumor cells or luteinizing hormone releasing hormone receptor (LHRHR or GNRHR) such as GNRHR1 on tumor cells.
  • SSTR somatostatin receptor
  • LHRHR or GNRHR luteinizing hormone releasing hormone receptor
  • the tumor cell binding moiety binds to a cell surface protein selected from the group consisting of CD20, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), and CD 19.
  • CD 19 binding agents that may be used as a tumor cell binding moiety in the conjugates include any CD 19 binding agent disclosed in Dreier et al. J Immunol , vol.170:4397 (2003)), in Klinger et al. ⁇ Blood, vol.119:6226 (2012)), or blinatumomab, a bispecific single-chain antibody targeting CD3 and CD19 antigen disclosed in Topp et al.
  • Non-limiting examples of CD20 binding agents include anti-CD20/CD3 T cell-dependent bispecific antibody disclosed in Sun et al. (Sci TranslMed., vol.7:287 (2015)) or anti-CD3 x anti-CD20 bispecific antibody disclosed in Gall et al. (Exp Hematol, vol.33(4):452 (2005)).
  • Non-limiting examples of CEA binding agents include CEA/CD3 -bispecific T cell-engaging (BiTE) antibody disclosed in Osada et al. (Cancer Immunol Immunother., vol.64(6):677 (2015)).
  • Non-limiting examples of EpCAM binding agents include EpCAM/CD3-bispecific T-cell engaging antibody MT110 disclosed in Cioffi et al. (Clin. Cancer Res., vol. l8(2):465 (2012)).
  • the targeting moiety is a protein scaffold.
  • the protein scaffold may be a non-antibody-derived protein scaffold, wherein the protein scaffold is based on nonantibody binding proteins.
  • the protein scaffold may be based on engineered Kunitz domains of human serine protease inhibitors (e.g., LAC1 -D1), DARPins (designed ankyrin repeat domains), avimers created from multimerized low-density lipoprotein receptor class A (LDLR-A), anticalins derived from lipocalins, knottins constructed from cysteine-rich knottin peptides, affibodies that are based on the Z-domain of staphylococcal protein A, adnectins or monobodies and pronectins based on the 10 th or 14 th extracellular domain of human fibronectin III, Fynomers derived from SH3 domains of human Fyn tyrosine kinase, or nanofitins (formerly A).
  • the protein scaffold may be based on a fibronectin domain.
  • the protein scaffold may be based on fibronectin type III (FN3) repeat protein.
  • the protein scaffold may be based on a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). Any protein scaffold based on a fibronectin domain disclosed in US Pat. No. 8569227 to Jacobs et al, the content of which is incorporated herein by reference in its entirety; may be used as a targeting moiety of the conjugate of the invention.
  • the protein scaffold may be any protein scaffold disclosed in Mintz and Crea, BioProcess, vol.1 1(2):40-48 (2013), the contents of which are incorporated herein by reference in their entirety. Any of the protein scaffolds disclosed in Tables 2-4 of Mintz and Crea may be used as a targeting moiety of the conjugate of the invention.
  • the targeting moiety is an arginylglycylaspartic acid (RGD) peptide, a tripeptide composed of L-arginine, glucine and L-aspartic acid, which is a common cell targeting element for cellular attachment via integrins.
  • RGD arginylglycylaspartic acid
  • a targeting moiety may be an antibody that specifically binds to a TAA and/or an antigenic peptide (epitope).
  • an antibody fragment e.g., an Fc fragment of an antibody
  • an antibody fragment may be used for the same purpose.
  • antibodies may be specific to a ubiquitous antigenic site on various cancers. Many studies have revealed that cancer cells share certain common characteristics. Many types of human cancer cells are characterized by substantial abnormalities in the glycosylation patterns of their cell-surface proteins and lipids (e.g., Hakomori et. al, 1996, Cancer Res. 56:5309-18; and Springer et al, 1997, JMol Med 75 :594-602). These differences have led to the identification of antigenic determinants on cancer cells. Natural IgM antibodies to these epitopes are present in the circulation and can be used as a targeting moiety of a conjugate of the present invention.
  • the antibody targeting moiety may be connected to one or more components of the complement system (or other cytotoxic agents) to induce complement mediated tumor cell lysis.
  • a conjugate may have a formula of (one or more cytotoxic agents)-linker -mAb.
  • the targeting moiety is an antibody mimetic such as a monobody, e.g., an ADNECTINTM (Bristol-Myers Squibb, New York, New York) , an Affibody® (Affibody AB, Sweden), Affilin, nanofitin (affitin, such as those described in WO 2012/085861, an AnticalinTM, an avimers (avidity multimers), a DARPinTM, a FynomerTM, CentyrinTM, and a Kunitz domain peptide.
  • ADNECTINTM Bristol-Myers Squibb, New York, New York
  • Affibody® Affibody AB, Sweden
  • Affilin nanofitin
  • affitin such as those described in WO 2012/085861
  • an AnticalinTM an avimers (avidity multimers)
  • DARPinTM a FynomerTM
  • CentyrinTM CentyrinTM
  • the targeting moiety X may be an aptide or bipodal peptide.
  • X may be any D-Aptamer-Like Peptide (D-Aptide) or retro-inverso Aptide which specifically binds to a target comprising: (a) a structure stabilizing region comprising parallel, antiparallel or parallel and antiparallel D-amino acid strands with interstrand noncovalent bonds; and (b) a target binding region I and a target binding region II comprising randomly selected n and m D-amino acids, respectively, and coupled to both ends of the structure stabilizing region, as disclosed in US Pat. Application No.
  • X may be any bipodal peptide binder (BPB) comprising a structure stabilizing region of parallel or antiparallel amino acid strands or a combination of these strands to induce interstrand non-covalent bonds, and target binding regions I and II, each binding to each of both termini of the structure stabilizing region, as disclosed in US Pat. Application No. 20120321697 to Jon et al, the content of which is incorporated herein by reference in its entirety.
  • BBP bipodal peptide binder
  • X may be an intracellular targeting bipodal - peptide binder specifically binding to an intracellular target molecule, comprising: (a) a structure-stabilizing region comprising a parallel amino acid strand, an antiparallel amino acid strand or parallel and antiparallel amino acid strands to induce interstrand non-covalent bonds; (b) target binding regions I and II each binding to each of both termini of the structure-stabilizing region, wherein the number of amino acid residues of the target binding region I is n and the number of amino acid residues of the target binding region II is m; and (c) a cell-penetrating peptide (CPP) linked to the structure-stabilizing region, the target binding region I or the target binding region II, as disclosed in US Pat. Application No.
  • CPP cell-penetrating peptide
  • X may be any bipodal peptide binder comprising a ⁇ -hairpin motif or a leucine- zipper motif as a structure stabilizing region comprising two parallel amino acid strands or two antiparallel amino acid strands, and a target binding region I linked to one terminus of the first of the strands of the structure stabilizing region, and a target binding region II linked to the terminus of the second of the strands of the structure stabilizing region, as disclosed in US Pat. Application No. 20110152500 to Jon et al, the content of which is incorporated herein by reference in its entirety.
  • X may be any bipodal peptide binder targeting KPI as disclosed in WO2014017743 to Jon et al, any bipodal peptide binder targeting cytokine as disclosed in WO2011132939 to Jon et al, any bipodal peptide binder targeting transcription factor as disclosed in WO201132941 to Jon et al., any bipodal peptide binder targeting G protein-coupled receptor as disclosed in WO2011132938 to Jon et al, any bipodal peptide binder targeting receptor tyrosine kinase as disclosed in WO2011132940 to Jon et al, the content of each of which is incorporated herein by reference in their entirety.
  • X may also be bipodal peptide binders targeting cluster differentiation (CD7) or an ion channel.
  • the targeting moiety is a stabilized peptide.
  • Intramolecular crosslinkers are used to maintain the peptide in the desired configuration, for example using disulfide bonds, amide bonds, or carbon-carbon bonds to link amino acid side chains. Such peptides which are conformationally stabilized by means of intramolecular cross-linkers are sometimes referred to as "stapled" peptides.
  • the cross-linkers connect at least two amino acids of the peptide.
  • the cross-linkers may comprise at least 5, 6, 7, 8, 9, 10, 11, or 12 consecutive carbon-carbon bonds.
  • the cross-linkers may comprise at least 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Stapled peptides may penetrate cell membranes and bind to an intracellular receptor.
  • the stapled peptide is a cross-linked alpha-helical polypeptide comprising a crosslinker wherein a hydrogen atom attached to an a-carbon atom of an amino acid of the peptide is replaced with a substituent of formula R-, wherein R- is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, as disclosed in US 20140323701 to Nash et al, the contents of which are incorporated herein by reference in their entirety.
  • the stapled peptides have improved in vivo half life such as any stapled peptide disclosed in US 20100298201 to Nash et al, the contents of which are incorporated herein by reference in their entirety.
  • the tumor cell binding moiety may be any stapled peptide disclosed in US 9175045 to Nash et al, the contents of which are incorporated herein by reference in their entirety, wherein the stapled peptide possesses reduced affinity to serum proteins while still remaining sufficient affinity to cell membranes.
  • the cross-linker of the stapled peptide links the a-positions of at least two amino acids, such as any stapled peptide disclosed in US 9175047 to Nash et al, the contents of which are incorporated herein by reference in their entirety.
  • the tumor cell binding moiety comprise any stapled peptide disclosed in US 8927500 to Guerlavais et al., the contents of which are incorporated herein by reference in their entirety, wherein the stapled peptide has homology to p53 protein and can bind to the MDM2 and/or MDMX proteins.
  • the stapled peptide generates a reduced antibody response.
  • any stapled peptide disclosed in US 8808694 to Nash et al, the contents of which are incorporated herein by reference in their entirety, may be used as a tumor cell binding moiety.
  • the stapled peptide may be any polypeptide with optimized protease stability disclosed in US 201 10223149 to Nash et al, the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety is a nanofitin® (Affilogic).
  • Nanofitin refers to a single-chain antibody mimic that are much smaller than antibodies. Nanofitins are small and stable, lack disulfide bridges, and can be produced at high levels. The molecular weight of nanofitins are below l OKDa, preferably around 7KDa. Because of their small size and short half-life, nanofitins may both accumulate specifically at the site of the tumor and be cleared from the serum rapidly, therefore reducing off-target toxicity compared to long lasting antibodies. Conjugates comprise nanofitins may deliver an active agent deeper into a tumor. Nanofitins may bind intracellular targets and affect intracellular protein-protein interaction.
  • the targeting moiety may be a bispecific T-cell engagers, an aptamer such as RNA, DNA or an artificial nucleic acid; a small molecule; a carbohydrate such as mannose, galactose or arabinose; a lipid, a vitamin such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid (folate), riboflavin, biotin, vitamin B 12, vitamin A, E, and K.
  • an aptamer such as RNA, DNA or an artificial nucleic acid
  • a small molecule such as mannose, galactose or arabinose
  • a lipid a vitamin such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid (folate), riboflavin, biotin, vitamin
  • the targeting moiety may comprise a nucleic acid targeting moiety.
  • a nucleic acid targeting moiety is any nucleic acid that binds to an organ, tissue, cell, or a component associated therewith such as extracellular matrix component, and intracellular compartment.
  • the targeting moiety may be an aptamer, which is generally an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that binds to a particular target, such as a polypeptide.
  • the targeting moiety may be an aptamer that targets to an immune cell (e.g., dendritic cells).
  • Aptamers may be generated from libraries of single-stranded nucleic acids against different molecules via CELL-SELEX method in which whole living cells (e.g., dendritic cells) are used as targets for the aptamers (Ganji et al, Aptamers: new arrows to target dendritic cells, J Drug Target. 2015, 7: 1 -12).
  • the targeting moiety may be a non-immunoreactive ligand.
  • the non-immunoreactive ligand may be insulin, insulin-like growth factors I and II, lectins, apoprotein from low density lipoprotein, etc. as disclosed in US 20140031535 to Jeffrey, the content of which is incorporated herein by reference in its entirety.
  • Any protein or peptide comprising a lectin disclosed in WO2013181454 to Radin, the content of which is incorporated herein by reference in its entirety, may be used as a targeting moiety.
  • targeting moieties may be Lymph Node-targeting nanoparticle (NP)-conjugates (Jeanbart et al, Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes. Cancer Immunol Res., 2014, 2(5): 436-437; the content of which is incorporated by reference in its entirety.
  • NP Lymph Node-targeting nanoparticle
  • the conjugate may have a terminal half-life of longer than about 72 hours and a targeting moiety may be selected from Table 1 or 2 of US 20130165389 to Schellenberger et al, the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be an antibody targeting delta-like protein 3 (DLL3) in disease tissues such as lung cancer, pancreatic cancer, skin cancer, etc., as disclosed in WO2014125273 to Hudson, the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety may also any targeting moiety in WO2007137170 to Smith, the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety binds to glypican-3 (GPC-3) and directs the conjugate to cells expressing GPC-3, such as hepatocellular carcinoma cells.
  • the targeting moiety may be a modified viral surface protein or fragments thereof.
  • the targeting moiety may be an antigen recognition domain/sequence of TCR molecules.
  • the nature of antigen recognition of such moieties will bind to an antigen-MHC molecule complex on the surface of cells, therefore deliver an active payload linked to the targeting moieties through a linker in the conjugate to the tumor cells.
  • targeting moieties may be derived from the binding domains of the MHC class I and II molecules, for example, the a3 domain of the a chain of the MHC class I molecule.
  • the a3 domain in the MHC class I molecule can specifically bind to CD8 on T cells, and the binding between CD8 and the a3 domain may deliver tumor antigen payloads near to the surface of T cells and activate TCR to bind the tumor antigens.
  • the targeting moiety may be the ⁇ 2 domain of the MHC class II molecules.
  • the targeting moiety may be a cell binding element such as a ligand which binds to a cell surface receptor.
  • the cell binding element may be selected from the group consisting of a Fc fragment, a toxin cell binding domain, a cytokine, a chemokine, a small peptide and an antibody.
  • the cytokines, chomekines and other immunomodulatory molecules are ligands of cell receptors on certain types of immune cells such as APCs (e.g., DCs), T cells, B cells, NK cells and macrophages.
  • targeting moieties may be used to deliver antigens to APCs (Frenz et al, Antigen presenting cell selective drug delivery by gly can-decorated
  • nanocarriers Eur J Pharm Biopharm, 2015, Feb 19, pii: S0939-6411), such as DEC-205 antibody as targeting moieties for targeted delivery of antigens to APCs.
  • targeting moieties may be a single-chain antibody mimic that are much smaller than antibodies such as nanofitin® (Affilogic) disclosed in copending US Application No. 62/308,908, or peptides which are conformationally stabilized by means of intramolecular cross-linkers referred to as "stapled” peptides disclosed in copending US Application No. 62/291 ,212, the contents of each of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be a targeting moiety complex comprising a target binding moiety (TBM) and a masking moiety (MM).
  • TBM target binding moiety
  • MM masking moiety
  • MM may be attached to TBM directly, via a non-cleavable moiety, or via a cleavable moiety (CM). In some other embodiments, MM is bound to the payload or the linker of the conjugate directly, via a non-cleavable moiety, or via a cleavable moiety (CM).
  • TBM may be any targeting moiety discussed above including small molecules, peptides or derivatives, an antibody or a fragment thereof.
  • TBM may be a peptide comprising between 5 to 50 amino acids, between 10 to 40 amino acids, or between 20 to 30 amino acids.
  • TBM may be small molecules.
  • the binding of TBM to its target is inhibited or hindered by MM.
  • the binding may be sterically hindered by the presence of MM or may be inhibited by the charge of MM. Leaving of MM upon cleavage of CM, a conformation change, or a chemical transformation may unmask TBM.
  • the masking/unmasking process may be reversible or irreversible.
  • TBM In one example wherein TBM is attached to MM with a CM, TBM might be less accessible to its target when CM is uncleaved. Upon cleavage of CM, MM no longer interferes with the binding of the targeting moiety to its target, thereby activating the conjugates of the present invention. The cleavable moiety prevents binding of the conjugates of the present invention at nontreatment sites. Such conjugates can further provide improved biodistribution characteristics.
  • MM may be selected from a plurality of polypeptides based on its ability to inhibit binding of the TBM to the target in an uncleaved state and allow binding of the TBM to the target in a cleaved state.
  • CM may locate between TBM and MM in the targeting moiety complex, or may locate within MM.
  • CM may be cleaved by an enzyme such as protease.
  • CM may comprise a peptide that may be a substrate for an enzyme selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM 10, AD AMI 2, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase- 8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
  • CM may comprise a protease substrate such as a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate (e.g., a substrate of MMP- 1, MMP-2, MMP-9, or MMP-14).
  • MMP matrix metalloprotease
  • CM may be cleaved by a reducing agent capable of reducing a disulfide bond between a cysteine-cysteine pair.
  • CM may comprise a cysteine-cysteine pair capable of forming a reducible disulfide bond.
  • Reducing agents of particular interest include cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate.
  • cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate.
  • the targeting moiety complex may be any activatable binding polypeptides (ABPs) disclosed in US9169321 to Daugherty et al. (CytomX), the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety complex may be an enzyme activatable binding polypeptide (ABP) that binds CTLA-4, VEGF, or VCAM-1.
  • the targeting moiety complex may be an activatable binding polypeptide (ABP) that binds epidermal growth factor disclosed in US 9120853 to Lowman et al, an ABP that binds Jagged 1 or Jagged 2 disclosed in US9127053 to West et al., activatable anti-CD3 antibodies disclosedin WO2016014974 to Irving et al, activatable antibodies that bind to interleukin-6 receptor (IL6R) disclosed in WO2014052462 to West et al, activatable proproteins disclosed in US20150203559 to Stagliano et al, any modified antibody or activatable antibody disclosed in
  • ABSP activatable binding polypeptide
  • the targeting moiety may be a targeting moiety complex comprising a target binding moiety (TBM) and a photocleavable moiety.
  • TBM target binding moiety
  • TBM may be any targeting moiety discussed above including small molecules, peptides or derivatives, an antibody or a fragment thereof.
  • a "photocleavable moiety” means any agent attached to the antibody which can be removed on exposure to electromagnetic energy such as light energy of any desired vanety whether visible, UV, X-ray or the like (e g microwave).
  • the photocleavable moiety may be a reagent which couples to hydroxy or amino residues present in TBM.
  • phosgene, diphosgene, DCCI or the like may be used to generate photocleavable esters, amides, carbonates and the like from a wide range of alcohols.
  • substituted arylalkanols are employed, particularly nitorphenyl methyl alcohol, 1-nitrophenylethan-l-ol and substituted analogues.
  • the photocleavable moiety may be located at or about the binding site of TBM.
  • the targeting moiety complex may comprise any photocleavable moiety disclosed in WO 1996034892 to Self et al., the contents of which are incorporated herein by reference in their entirety.
  • TBM may be an antibody component that retain the active site and bind to a tumor cell marker.
  • TBM may also be any antibody component made against suitable cells such as T-cells, cytotoxic T-cell clones, cytotoxic T-cells and activated peripheral blood lymphocytes, CD3+ lymphocytes, CD 16+ lymphocytes, Fc gamma Rl 11, the low affinity Fc gamma receptor for polymorphonuclear leucocytes, macrophages and large granular lymphocytes, B-lymphocyte markers, myeloid cells, T Lymphocyte CD2, CD3, CD4, CD8, dengue virus, lymphokine activated killer (LAK) cells, NK cells or monocytes.
  • TBM may be a monoclonal antibody anti-CD-3 OKT3 against T-cells, or a monoclonal antibody that binds to tumor antigen carcinoembrionic antigen (CEA).
  • CEA tumor antigen carcinoembrionic antigen
  • the targeting moiety or moieties of the conjugate are present at a predetermined molar weight percentage from about 1 % to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%.
  • the amount of targeting moieties of the conjugate may also be expressed in terms of proportion to the active agent(s), for example, in a ratio of ligand to active agent of about 10: 1 , 9: 1, 8: 1 , 7: 1, 6: 1, 5: 1 , 4: 1 , 3 : 1, 2: 1 , 1 : 1 , 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 : 8, 1 :9, or 1 : 10.
  • the conjugates of the present invention may further comprise at least one external linker connected to a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof, or comprise at least one external linker connected to a pharmacokinetic modulating unit.
  • modulating units may be cleavable linkers that allow release of the conjugates.
  • the conjugates may be separated from the protein or pharmacokinetic modulating units as needed.
  • the conjugates comprise at least one reacting group that reacts with a functional group on a protein or an engineered protein or
  • the reaction between the reacting group and the functional group may happen in vivo after administration or is performed prior to administration.
  • the protein may be a naturally occurring protein such as a serum or plasma protein, or a fragment thereof. Particular examples include thyroxine-binding protein, transthyretin, ⁇ -acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, ⁇ -2-macroglobulin, a lipoprotein, or fragments thereof.
  • the reaction between the reacting group and the functional group may be reversible.
  • the functional group is on human serum albumin (HSA or albumin) or its derivative/analog/mimic.
  • Albumin is the most abundant plasma protein (35-50 g/L in human serum) with a molecular weight of 66.5 KDa and an effective diameter of 7.2 nm (Kratz, J. of Controlled Release, vol.132: 171, (2008), the contents of which are incorporated herein by reference in their entirety). Albumin has a half-life of about 19 days. Albumin preferentially accumulates in malignant and inflamed tissues due to a leaky capillary and an absent or defective lymphatic drainage system. Albumin accumulates in tumors such as solid tumors also because albumin is a major energy and nutrition source for turmor growth.
  • the function group may be the cysteine-34 position of albumin that has an accessible free thiol group.
  • Reacting groups that react with a functional group on albumin or it derivative/analog/mimic may be selected from a disulfide group, a vinylcarbonyl group, a vinyl acetylene group, an aziridine group, an acetylene group or any of the following groups:
  • R 7 is CI, Br, F, mesylate, tosylate, 0-(4-nitrophenyl), O-pentafluorophenyl, and wherein optionally the activated disulfide group, the vinylcarbonyl group, the vinyl acetylene group, the aziridine group, and the acetylene group may be substituted.
  • the reacting group may also be any protein-binding moiety disclosed in US 9216228 to Kratz et al, the contents of which are incorporated herein by reference in their entirety, selected from the group consisting of a maleinimide group, a halogenacetamide group, a halogenacetate group, a pyridylthio group, a vinylcarbonyl group, an aziridine group, a disulfide group, a substituted or unsubstituted acetylene group, and a hydroxysuccinimide ester group.
  • the reacting group is a disulfide group.
  • the disulfide group undergoes an exchange with a thiol group on a protein or an engineered protein or a polymer or derivatives/analogs/mimics thereof, such as albumin, to form a disulfide between the conjugate and the protein or an engineered protein or a polymer or derivatives/analogs/mimics thereof.
  • the functional group is on transthyretin or its
  • Transthyretin is a 55 KDa serum protein that has an in vivo half-life of around 48 h.
  • Reacting groups that react with a functional group on transthyretin or it derivative/analog/mimic may be selected from AGI O (structure shown below) or its derivative disclosed by Penchala et al. Nature Chemical Biology, vol.1 1 :793, (2015) or formula (I), (II), (III) or (IV) (structures shown below) disclosed in US Pat. No. 5714142 to Blaney et al., the contents of each of which are incorporated herein by reference in their entirety.
  • Any transthyretin-selective ligand disclosed on pages 5-8 of Blaney et al. or their derivatives may be used as a reacting group, such as but not limited to, tetraiodothyroacetic
  • the reacting group may be any protein binding moiety may be any protein binding moiety disclosed in US 9216228 to Kratz, the contents of which are incorporated herein by reference in their entirety, such as a maleimide group, a
  • halogenacetamide group a halogenacetate group, a pyridylthio group, a vinylcarbonyl group, an aziridin group, a disulfide group, a substituted or unsubstituted acetylene group, and a hydroxy succinimide ester group.
  • the conjugates comprise at least one pharamacokinetic modulating unit.
  • the pharmacokinetic modulating unit may be a natural or synthetic protein or fragment thereof.
  • it may be a serum protein such as thyroxine- binding protein, transthyretin, ⁇ -acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, ⁇ -2-macroglobulin, a lipoprotein, or fragments thereof.
  • the pharmacokinetic modulating unit may also be a natural or synthetic polymer, such as polysialic acid unit, a hydroxy ethyl starch (HES) unit, or a polyethylene glycol (PEG) unit.
  • the pharmacokinetic modulating unit may be a particle, such as dendrimers, inorganic nanoparticles, organic nanoparticles, and liposomes.
  • the pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight of at least about 10 KDa, at least about 20 KDa, at least about 30 KDa, at least about 40 KDa or at least about 50 KDa.
  • the pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight between about 10 KDa and about 70 KDa.
  • the pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight between about 30 KDa and about 70 KDa, between about 40 KDa and about 70 KDa, between about 50 KDa and about 70 KDa, between about 60 KDa and about 70 KDa.
  • Particles comprising one or more conjugates can be polymeric particles, lipid particles, solid lipid particles, solid lipid nanoparticles, solid nanoparticles, inorganic particles, or combinations thereof (e.g., lipid stabilized polymeric particles).
  • the conjugates are substantially encapsulated or particularly encapsulated in the particles.
  • the conjugates are disposed on the surface of the particles. The conjugates may be attached to the surface of the particles with covalent bonds, or non-covalent interactions.
  • the conjugates of the present invention self-assemble into a particle.
  • the term "encapsulate” means to enclose, surround or encase. As it relates to the formulation of the conjugates of the invention, encapsulation may be substantial, complete or partial.
  • the term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.999% of conjugate of the invention may be enclosed, surrounded or encased within the particle.
  • Partially encapsulation means that less than 10, 10, 20, 30, 40 50 or less of the conjugate of the invention may be enclosed, surrounded or encased within the particle. For example, at least 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the particle. Encapsulation may be determined by any known method.
  • the particles are polymeric particles or contain a polymeric matrix.
  • the particles can contain any of the polymers described herein or derivatives or copolymers thereof.
  • the particles will generally contain one or more biocompatible polymers.
  • the polymers can be biodegradable polymers.
  • the polymers can be hydrophobic polymers, hydrophilic polymers, or amphiphilic polymers.
  • the particles contain one or more polymers having an additional targeting moiety attached thereto.
  • the particles are inorganic particles, such as but not limited to, gold nanoparticles and iron oxide
  • the size of the particles can be adjusted for the intended application.
  • the particles can be nanoparticles or microparticles.
  • the particle can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, or about 25 nm to about 250 nm.
  • the particle is a nanoparticle having a diameter from about 25 nm to about 250 nm.
  • the particle is a nanoparticle having a diameter from about 50 nm to about 150 nm.
  • the particle is a nanoparticle having a diameter from about 70 nm to about 130 nm.
  • the particle is a nanoparticle having a diameter of about 100 nm. It is understood by those in the art that a plurality of particles will have a range of sizes and the diameter is understood to be the median diameter of the particle size distribution.
  • Polydispersity index (PDI) of the particles may be ⁇ about 0.5, ⁇ about 0.2, or ⁇ about 0.1.
  • Drug loading may be > about 1%, > about 5%, > about 10%, or > out 20%.
  • Drug loading refers to the weight ratio of the conjugates of the invention and depends on maximum tolerated dose (MTD) of free drug conjugate.
  • Particle ⁇ -potential in 1/10 th PBS
  • Drug released in vitro from the particle at 2h may be less than about 60%, less than about 40%, or less than about 20%.
  • pharmacokinetics, plasma area under the curve (AUC) in a plot of concentration of drug in blood plasma against time may be at least 2 fold greater than free drug conjugate, at least 4 fold greater than free drug conjugate, at least 5 fold greater than free drug conjugate, at least 8 fold greater than free drug conjugate, or at least 10 fold greater than free drug conjugate.
  • Tumor PK PD of the particle may be at least 5 fold greater than free drug conjugate, at least 8 fold greater than free drug conjugate, at least 10 fold greater than free drug conjugate, or at least 15 fold greater than free drug conjugate.
  • the ratio of Cmax of the particle to Cmax of free drug conjugate may be at least about 2, at least about 4, at least about 5, or at least about 10.
  • Cmax refers to the maximum or peak serum concentration that a drug achieves in a specified compartment or test area of the body after the drug has been administrated and prior to the administration of a second dose.
  • the ratio of MTD of a particle to MTD of free drug conjugate may be at least about 0.5, at least about 1, at least about 2, or at least about 5. Efficacy in tumor models, e.g., TGI%, of a particle is better than free drug conjugate.
  • Toxicity of a particle is lower than free drug conjugate.
  • a particle may be a nanoparticle, i.e., the particle has a characteristic dimension of less than about 1 micrometer, where the characteristic dimension of a particle is the diameter of a perfect sphere having the same volume as the particle.
  • the plurality of particles can be characterized by an average diameter (e.g., the average diameter for the plurality of particles).
  • the diameter of the particles may have a Gaussian-type distribution.
  • the plurality of particles have an average diameter of less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 3 nm, or less than about 1 nm. In some embodiments, the particles have an average diameter of at least about 5 nm, at least about 10 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, or greater.
  • the plurality of the particles have an average diameter of about 10 nm, about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 50 nm and about 400 nm, between about 100 nm and about 300 nm, between about 150 nm and about 250 nm, between about 175 nm and about 225 nm, or the like.
  • the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 20 nm and about 400 nm, between about 30 nm and about 300 nm, between about 40 nm and about 200 nm, between about 50 nm and about 175 nm, between about 60 nm and about 150 nm, between about 70 nm and about 130 nm, or the like.
  • the average diameter can be between about 70 nm and 130 nm.
  • the plurality of particles have an average diameter between about 20 nm and about 220 nm, between about 30 nm and about 200 nm, between about 40 nm and about 180 nm, between about 50 nm and about 170 nm, between about 60 nm and about 150 nm, or between about 70 nm and about 130 nm.
  • the particles have a size of 40 to 120 nm with a zeta potential close to 0 mV at low to zero ionic strengths (1 to 10 mM), with zeta potential values between + 5 to - 5 mV, and a zero/neutral or a small -ve surface charge.
  • the particles of the invention may comprise more than one conjugates.
  • the conjugates may be different, e.g., comprising different payloads.
  • the particles of the invention may comprises conjugates having different PK values. Conjugates in the same particle are protected by the particle and are released at the same time. In some embodiments, linkers of the conjugates are cleaved under the same condition and payloads of the conjugates are released at the same time. In some
  • linkers of the conjugates are cleaved under different condistions and payloads of the conjugates are released sequentially.
  • the particles of the invention may comprise a first conjugate having immune stimulating agents as payloads and a second conjugate having antigens as payloads. The linkers of the first conjugate are cleaved before the linkers of the second conjugate, thereby releaving the immune stimulating agents and then the antigens.
  • the weight percentage of the conjugate in the particles is at least about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% such that the sum of the weight percentages of the components of the particles is 100%.
  • the weight percentage of the conjugate in the particles is from about 0.5% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the weight percentages of the components of the particles is 100%.
  • the particles of the invention may contain one or more polymers.
  • Polymers may contain one more of the following polyesters: homopolymers including gly colic acid units, referred to herein as "PGA”, and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA”, and caprolactone units, such as poly(8-caprolactone), collectively referred to herein as "PCL”; and copolymers including lactic acid and gly colic acid units, such as various forms of poly(lactic acid-co-gly colic acid) and poly(lactide-co- glycolide) characterized by the ratio of lactic acid:gly colic acid, collectively referred to herein as "PLGA”; and polyacrylates, and derivatives thereof.
  • PGA gly colic acid units
  • PLA poly-
  • Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers".
  • PEG polyethylene glycol
  • the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker.
  • the particles may contain one or more hydrophilic polymers.
  • Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-poly glutamic acid, poly-L- aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone);
  • the particles may contain one or more hydrophobic polymers.
  • suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(gly colic acid), and poly(lactic acid-co-gly colic acids); polyhydroxyalkanoates such as poly3- hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);
  • polyanhydrides poly(phosphazenes); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones);
  • poly(alkylene alkylates) hydrophobic poly ethers; polyurethanes; polyetheresters;
  • polyacetals polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals; polyphosphates;
  • polyhydroxyvalerates polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof.
  • the hydrophobic polymer is an aliphatic polyester. In some embodiments, the hydrophobic polymer is poly(lactic acid), poly(gly colic acid), or poly(lactic acid-co-gly colic acid).
  • the particles can contain one or more biodegradable polymers.
  • Biodegradable polymers can include polymers that are insoluble or sparingly soluble in water that are converted chemically or enzymatically in the body into water-soluble materials.
  • Biodegradable polymers can include soluble polymers crosslinked by hydolyzable cross- linking groups to render the crosslinked polymer insoluble or sparingly soluble in water.
  • Biodegradable polymers in the particle can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,
  • polyvinylpyrrolidone polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose such as methyl cellulose and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy -propyl methyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, polymers of acrylic and methacrylic esters such as poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly
  • biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof.
  • the particle contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(gly colic acid), and poly(lactic-co-gly colic acid).
  • the particles can contain one or more amphiphilic polymers.
  • Amphiphilic polymers can be polymers containing a hydrophobic polymer block and a hydrophilic polymer block.
  • the hydrophobic polymer block can contain one or more of the hydrophobic polymers above or a derivative or copolymer thereof.
  • the hydrophilic polymer block can contain one or more of the hydrophilic polymers above or a derivative or copolymer thereof.
  • amphiphilic polymer is a di-block polymer containing a hydrophobic end formed from a hydrophobic polymer and a hydrophilic end formed of a hydrophilic polymer.
  • a moiety can be attached to the hydrophobic end, to the hydrophilic end, or both.
  • the particle can contain two or more amphiphilic polymers.
  • the particles may contain one or more lipids or amphiphilic compounds.
  • the particles can be liposomes, lipid micelles, solid lipid particles, or lipid-stabilized polymeric particles.
  • the lipid particle can be made from one or a mixture of different lipids.
  • Lipid particles are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH.
  • the lipid particle is preferably made from one or more biocompatible lipids.
  • the lipid particles may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH.
  • the particle can be a lipid micelle.
  • Lipid micelles for drug delivery are known in the art.
  • Lipid micelles can be formed, for instance, as a water-in-oil emulsion with a lipid surfactant.
  • An emulsion is a blend of two immiscible phases wherein a surfactant is added to stabilize the dispersed droplets.
  • the lipid micelle is a microemulsion.
  • a microemulsion is a thermodynamically stable system composed of at least water, oil and a lipid surfactant producing a transparent and thermodynamically stable system whose droplet size is less than 1 micron, from about 10 nm to about 500 nm, or from about 10 nm to about 250 nm.
  • Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents.
  • the particle can be a liposome.
  • Liposomes are small vesicles composed of an aqueous medium surrounded by lipids arranged in spherical bilayers. Liposomes can be classified as small unilamellar vesicles, large unilamellar vesicles, or multi-lamellar vesicles. Multi-lamellar liposomes contain multiple concentric lipid bilayers. Liposomes can be used to encapsulate agents, by trapping hydrophilic agents in the aqueous interior or between bilayers, or by trapping hydrophobic agents within the bilayer.
  • the lipid micelles and liposomes typically have an aqueous center.
  • the aqueous center can contain water or a mixture of water and alcohol.
  • Suitable alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as w-butanol, isobutanol, seobutanol, fert-butanol, pentanol (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1 -heptanol, 2-heptanol, 3-heptanol and 4-heptanol) or octanol (such as 1-octanol) or a combination thereof.
  • the particle can be a solid lipid particle.
  • Solid lipid particles present an alternative to the colloidal micelles and liposomes.
  • Solid lipid particles are typically submicron in size, i.e. from about 10 nm to about 1 micron, from 10 nm to about 500 nm, or from 10 nm to about 250 nm.
  • Solid lipid particles are formed of lipids that are solids at room temperature. They are derived from oil-in-water emulsions, by replacing the liquid oil by a solid lipid.
  • Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids.
  • Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including 1 ,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;
  • PC phosphatidylcholine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1- ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl- sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC).
  • DOPE dioleylphosphoethanolamine
  • DHPE 1,2-dihexadecylphosphoethanolamine
  • the lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated l,2-diacyl-OT-glycero-3-phosphocholines, l-acyl-2-acyl-s??-glycero- 3-phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids.
  • tissue derived L-a-phosphatidyl egg yolk, heart, brain, liver, soybean
  • synthetic e.g., saturated and unsaturated l,2-diacyl-OT-glycero-3-phosphocholines, l-acyl-2-acyl-s??-glycero- 3-phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine
  • Suitable cationic lipids include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt.
  • Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-).
  • Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[l-(2,3- dioloyloxy)propyl]-N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dialkyloxy-3-dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 - N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi); 2,3-dioleoyloxy-N-(2- (sper
  • the cationic lipids can be l-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, for example, l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2-(hexadecanoyloxy)ethyl]-2- pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM).
  • DOTIM hydroxyethyl
  • DPTIM pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride
  • the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3- dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxy
  • Suitable solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and mono-, di-, and triglycerides of higher saturated fatty acids.
  • Solid lipids can include aliphatic alcohols having 10-40, preferably 12- 30 carbon atoms, such as cetostearyl alcohol.
  • Solid lipids can include higher fatty acids of 10- 40, preferably 12-30 carbon atoms, such as stearic acid, palmitic acid, decanoic acid, and behenic acid.
  • Solid lipids can include glycerides, including monoglycerides, diglycerides, and triglycerides, of higher saturated fatty acids having 10-40, preferably 12-30 carbon atoms, such as glyceryl monostearate, glycerol behenate, glycerol palmitostearate, glycerol trilaurate, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil.
  • Suitable solid lipids can include cetyl palmitate, beeswax, or cyclodextrin.
  • Amphiphilic compounds include, but are not limited to, phospholipids, such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of between 0.01-60 (weight lipid/w polymer), for example, between 0.1-30 (weight lipid/w polymer).
  • Phospholipids which may be used include, but are not limited to, phosphatidic acids, phosphatidyl cholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines,
  • phospholipids include, but are not limited to, phosphatidylcholines such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine
  • dilauroylphosphatidylcholine dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines such as
  • dioleoylphosphatidylethanolamine or 1 -hexadecyl-2-palmitoylgly cerophos-phoethanolamine may also be used.
  • the particles contain one or more immunological conjugates as described above.
  • the conjugates can be present on the interior of the particle, on the exterior of the particle, or both.
  • immunological conjugates refers ot any conjugates that can modulate an immune response in a subject, in particular, an anti-cancer immune response.
  • the conjugates may comprise any combination of the payloads, linkers and targeting moieties as described in the previous sections.
  • the particles may comprise hydrophobic ion-pairing complexes or hydrophobic ioin-pairs formed by one or more conjugates described above and counterions.
  • Hydrophobic ion-pairing is the interaction between a pair of oppositely charged ions held together by Coulombic attraction.
  • HIP refers to the interaction between the conjugate of the present invention and its counterions, wherein the counterion is not H + or HO " ions.
  • Hydrophobic ion-pairing complex or hydrophobic ion-pair refers to the complex formed by the conjugate of the present invention and its counterions.
  • the counterions are hydrophobic.
  • the counterions are provided by a hydrophobic acid or a salt of a hydrophobic acid.
  • the counterions are provided by bile acids or salts, fatty acids or salts, lipids, or amino acids.
  • the counterions are negatively charged (anionic). Non- limited examples of negative charged counterions include the counterions sodium
  • Non-limited examples of positively charged counterions include 1,2-dioleoy 1-3 -trimethylammonium- propane (chloride salt) (DOTAP), cetrimonium bromide (CTAB), quaternary ammonium salt didodecyl dimethylammonium bromide (DMAB) or Didodecyldimethylammonium bromide (DDAB).
  • DOTAP 1,2-dioleoy 1-3 -trimethylammonium- propane
  • CTAB cetrimonium bromide
  • DMAB quaternary ammonium salt didodecyl dimethylammonium bromide
  • DDAB Didodecyldimethylammonium bromide
  • HIP may increase the hydrophobicity and/or lipophilicity of the conjugate of the present invention.
  • increasing the hydrophobicity and/or lipophilicity of the conjugate of the present invention may be beneficial for particle formulations and may provide higher solubility of the conjugate of the present invention in organic solvents.
  • particle formulations that include HIP pairs have improved formulation properties, such as drug loading and/or release profile.
  • slow release of the conjugate of the invention from the particles may occur, due to a decrease in the conjugate's solubility in aqueous solution.
  • complexing the conjugate with large hydrophobic counterions may slow diffusion of the conjugate within a polymeric matrix.
  • HIP occurs without covalent conjuatation of the counterion to the conjugate of the present invention.
  • the strength of HIP may impact the drug load and release rate of the particles of the invention.
  • the strength of the HIP may be increased by increasing the magnitude of the difference between the pKa of the conjugate of the present invention and the pKa of the agent providing the counterion.
  • the conditions for ion pair formation may impact the drug load and release rate of the particles of the invention.
  • any suitable hydrophobic acid or a combination thereof may form a HIP pair with the conjugate of the present invention.
  • the hydrophobic acid may be a carboxylic acid (such as but not limited to a monocarboxylic acid, dicarboxylic acid, tricarboxylic acid), a sulfinic acid, a sulfenic acid, or a sulfonic acid.
  • a salt of a suitable hydrophobic acid or a combination thereof may be used to form a HIP pair with the conjugate of the present invention. Examples of hydrophobic acids, saturated fatty acids, unsaturated fatty acids, aromatic acids, bile acid, polyelectrolyte, their dissociation constant in water (pKa) and logP values were disclosed in
  • WO2014/043, 625 the content of which is incorporated herein by reference in its entirety.
  • the strength of the hydrophobic acid, the difference between the pKa of the hydrophobic acid and the pKa of the conjuagate of the present invention, logP of the hydrophobic acid, the phase transition temperature of the hydrophobic acid, the molar ratio of the hydrophobic acid to the conjugate of the present invention, and the concentration of the hydrophobic acid were also disclosed in WO2014/043,625, the content of which is incorporated herein by reference in its entirety.
  • particles of the present invention comprising a HIP complex and/or prepared by a process that provides a counterion to form HIP complex with the conjugate may have a highter drug loading than particles without a HIP complex or prepared by a process that does not provide any counterion to form HIP complex with the conjugate.
  • drug loading may increase 50%, 100%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
  • the particles of the invention may retain the conjugate for at least about 1 minute, at least about 15 minutes, at least about 1 hour, when placed in a phosphate buffer solution at 37°C
  • the particles may further comprise one or more immunologic adjuvants.
  • immunologic adjuvant refers to a compound or a mixture of compounds that acts to accelerate, prolong, enhance or modify immune responses when used in conjugation with an immunogen (e.g., neoantigens).
  • Adjuvant may be non-immunogenic when administered to a host alone, but that augments the host's immune response to another antigen when administered conjointly with that antigen.
  • adjuvant and “immunologic adjuvant” are used interchangeably in the present invention.
  • Adjuvant- mediated enhancement and/or extension of the duration of the immune response can be assessed by any method known in the art including without limitation one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant/antigen combination versus those produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines.
  • Adjuvants may be aluminium based adjuvants including but not limiting to aluminium hydroxide and aluminium phosphate; saponins such as steroid saponins and triterpenold saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines and routes of administrations.
  • Adjuvants may include, but are not limited to, alpha glucose bearing
  • glycosphingolipid compounds disclosed by Chen et al US Patent publication NO.
  • Those compounds when added into the present particles in combination with conjugates of the present invention can elevate invariant natural killer T (iNKT) cells and increases cytokine and/or chemokine production, where the cytokine production is sufficient to transactivate downstream immune cells including dendritic cells, natural killer cells, B cells, CD+4 T and CD8+ T cells.
  • iNKT invariant natural killer T
  • adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation.
  • Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs).
  • APCs antigen presenting cells
  • DCs dendritic cells
  • Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity.
  • adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation.
  • This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures (Powell et al, Clin Exp. Vaccine Res., Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. 2015, 4(l):23-45).
  • the partilcles further comprise pidotimod as an adjuvant.
  • the particles can contain one or more additional active agents in addition to those in the conjugates.
  • the additional active agents can be therapeutic, prophylactic, diagnostic, or nutritional agents as listed above.
  • the additional active agents can be present in any amount, e.g. from about 1% to about 90%, from about 1% to about 50%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 10%, or from about 5% to about 10% (w/w) based upon the weight of the particle.
  • the agents are incorporated in a about 1% to about 10% loading w/w.
  • the particles can contain one or more targeting moieties targeting the particle to a specific organ, tissue, cell type, or subcellular compartment in addition to the targeting moieties of the conjugate.
  • the additional targeting moieties can be present on the surface of the particle, on the interior of the particle, or both.
  • the additional targeting moieties can be immobilized on the surface of the particle, e.g., can be covalently attached to polymer or lipid in the particle.
  • the additional targeting moieties are covalently attached to an amphiphilic polymer or a lipid such that the targeting moieties are oriented on the surface of the particle.
  • conjugates, particles of the present invention may be formulated as vaccines, provided as liquid suspensions or as freeze-dried products.
  • suitable liquid preparations may include, but are not limited to, isotonic aqueous solutions, suspensions, emulsions, or viscous compositions that are buffered to a selected pH.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • active ingredient refers to any chemical and biological substance that has a physiological effect in human or in animals, when exposed to it.
  • the active ingredient in the formulations may be any conjugates and particles as discussed herein above.
  • a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1 -30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the conjugates or particles of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo.
  • excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and
  • Excipients of the present invention may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof.
  • formulations of the invention may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compounds.
  • the conjugates or particles of the present invention are formulated in aqueous formulations such as pH 7.4 phosphate-buffered formulation, or pH 6.2 citrate-buffered formulation; formulations for lyophilization such as pH 6.2 citrate- buffered formulation with 3% mannitol, pH 6.2 citrate-buffered formulation with 4% mannitol/1% sucrose; or a formulation prepared by the process disclosed in US Pat. No. 8883737 to Reddy et al. (Endocyte), the contents of which are incorporated herein by reference in their entirety.
  • aqueous formulations such as pH 7.4 phosphate-buffered formulation, or pH 6.2 citrate-buffered formulation
  • formulations for lyophilization such as pH 6.2 citrate- buffered formulation with 3% mannitol, pH 6.2 citrate-buffered formulation with 4% mannitol/1% sucrose
  • the conjugates or particles of the present invention targets folate receptors and are formulated in liposomes prepared following methods by Leamon et al. in Bioconjugate Chemistry, vol.14 738-747 (2003), the contents of which are incorporated herein by reference in their entirety. Briefly, folate-targeted liposomes will consist of 40 mole % cholesterol, either 4 mole % or 6 mole % polyethylene glycol (Mr ⁇ 2000)-derivatized phosphatidylethanolamine (PEG2000-PE, Nek tar.
  • Liposomes will be extruded 10 times through a 50 nm pore size polycarbonate membrane using a high-pressure extruder. Similarly, liposomes not targeting folate receptors may be prepared identically with the absence of folate-cysteine-PEG3400- PE.
  • the conjugates or particles of the present invention are formulated in parenteral dosage forms including but limited to aqueous solutions of the conjugates or particles, in an isotonic saline, 5% glucose or other pharmaceutically acceptable liquid carriers such as liquid alcohols, glycols, esters, and amides, as disclosed in US 7910594 to Vlahov et al. (Endocyte), the contents of which are incorporated herein by reference in their entirety.
  • the parenteral dosage form may be in the form of a reconstitutable lyophilizate comprising the dose of the conjugates or particles.
  • Any prolonged release dosage forms known in the art can be utilized such as, for example, the biodegradable carbohydrate matrices described in U. S. Pat. Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of which are incorporated herein by reference, or, alternatively, a slow pump (e.g., an osmotic pump) can be used.
  • the parenteral formulations are aqueous solutions containing carriers or excipients such as salts, carbohydrates and buffering agents (e.g.,at a pH of from 3 to 9).
  • the conjugates or particles of the present invention may be formulated as a sterile non-aqueous solution or as a dried form and may be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • a suitable vehicle such as sterile, pyrogen-free water.
  • the preparation of parenteral formulations under sterile conditions for example, by lyophilization under sterile conditions, may readily be accomplished using standard pharmaceutical techniques well- known to those skilled in the art.
  • the solubility of a conjugates or particles used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility -enhancing agents.
  • the conjugates or particles of the present invention may be prepared in an aqueous sterile liquid formulation comprising monobasic sodium phosphate monohydrate, dibasic disodium phosphate dihydrate, sodium chloride, potassium chloride and water for inj ection, as disclosed in US 20140140925 to Leamon et al, the contents of which are incorporated herein by reference in their entirety.
  • the conjugates or particles of the present invention may be formulated in an aqueous liquid of pH 7.4, phosphate buffered formulation for intravenous administration as disclosed in Example 23 of WO201 1014821 to Leamon et al. (Endocyte), the contents of which are incorporated herein by reference in their entirety.
  • the aqueous formulation needs to be stored in the frozen state to ensure its stability.
  • the conjugates or particles of the present invention are formulated for intravenous (IV) administration.
  • IV intravenous
  • the conjugates or particles may be formulated in an aqueous sterile liquid formulation of pH 7.4 phosphate buffered composition comprising sodium phosphate, monobasic monohydrate, disodium phosphate, dibasic dehydrate, sodium chloride, and water for injection.
  • the conjugates or particles may be formulated in pH 6.2 citrated-buffered formulation comprising trisodium citrate, dehydrate, citric acid and water for injection.
  • the conjugates or particles may be formulated with 3% mannitol in a pH 6.2 citrate-buffered formulation for lyophilization comprising trisodium citrate, dehydrate, citric acid and mannitol.
  • 3% mannitol may be replaced with 4% mannitol and 1 % sucrose.
  • the particles comprise biocompatible polymers.
  • the particles comprise about 0.2 to about 35 weight percent of a therapeutic agent; and about 10 to about 99 weight percent of a biocompatible polymer such as a diblock poly(lactic) acid-poly(ethylene)glycol as disclosed in US 20140356444 to Troiano et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.
  • BIND Therapeutics a diblock poly(lactic) acid-poly(ethylene)glycol as disclosed in US 20140356444 to Troiano et al.
  • Any therapeutically particle composition in US 8663700, 8652528, 8609142, 8293276 and 8420123 the contents of each of which are incorporated herein by reference in their entirety, may also be used.
  • the particles comprise a hydrophobic acid.
  • the particles comprise about 0.05 to about 30 weight percent of a substantially hydrophobic acid; about 0.2 to about 20 weight percent of a basic therapeutic agent having a protonatable nitrogen; wherein the pKa of the basic therapeutic agent is at least about 1.0 pKa units greater than the pKa of the hydrophobic acid; and about 50 to about 99.75 weight percent of a diblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblock poly(lactic acid-co-gly colic acid)-poly(ethylene)glycol copolymer, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly (ethylene)gly col as disclosed in
  • WO2014043625 to Figueiredo et al. BIND Therapeutics
  • the particles comprise a chemotherapeutic agent; a diblock copolymer of poly(ethylene)glycol and polylactic acid; and a ligand conjugate, as disclosed in US 20140235706 to Zale et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety. Any of the particle compositions in US 8603501, 8603500, 8603499, 8273363, 8246968, 20130172406 to Zale et al., may also be used. [00281] In some embodiments, the particles comprise a targeting moiety.
  • the particles may comprise about 1 to about 20 mole percent PLA-PEG-basement vascular membrane targeting peptide, wherein the targeting peptide comprises PLA having a number average molecular weight of about 15 to about 20 kDa and PEG having a number average molecular weight of about 4 to about 6 kDa; about 10 to about 25 weight percent anti-neointimal hyperplasia (NIH) agent; and about 50 to about 90 weight percent non- targeted poly -lactic acid-PEG, wherein the therapeutic particle is capable of releasing the anti-NIH agent to a basement vascular membrane of a blood vessel for at least about 8 hours when the therapeutic particle is placed in the blood vessel as disclosed in US 8563041 to Grayson et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.
  • the targeting peptide comprises PLA having a number average molecular weight of about 15 to about 20 kDa and PEG having a number average molecular weight of about 4 to about 6 kDa;
  • the particles comprise about 4 to about 25% by weight of an anti-cancer agent; about 40 to about 99% by weight of poly(D,L-lactic)acid- poly(ethylene)glycol copolymer; and about 0.2 to about 10 mole percent PLA-PEG-ligand; wherein the pharmaceutical aqueous suspension have a glass transition temperature between about 39 and 41°C, as disclosed in US 8518963 to Ali et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.
  • the particles comprise about 0.2 to about 35 weight percent of a therapeutic agent; about 10 to about 99 weight percent of a diblock poly (lactic) acid- poly(ethylene)glycol copolymer or a diblock poly(lactic)-co-poly (gly colic) acid- poly(ethylene)glycol copolymer; and about 0 to about 75 weight percent poly(lactic) acid or poly(lactic) acid-co-poly (gly colic) acid as disclosed in WO2012166923 to Zale et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.
  • the particles are long circulating and may be formulated in a biocompatible and injectable formulation.
  • the particles may be a sterile, biocompatible and injectable nanoparticle composition comprising a plurality of long circulating nanoparticles having a diameter of about 70 to about 130 nm, each of the plurality of the long circulating nanoparticles comprising about 70 to about 90 weight percent poly(lactic) acid-co-poly(ethylene) glycol, wherein the weight ratio of poly(lactic) acid to poly(ethylene) glycol is about 15 kDa/2 kDa to about 20 kDa/10 kDa, and a therapeutic agent encapsulated in the nanoparticles as disclosed in US 20140093579 to Zale et al.
  • a reconstituted lyophilized pharmaceutical composition suitable for parenteral administration comprising the particles of the present invention.
  • the reconstituted lyophilized pharmaceutical composition may comprise a 10-100 mg/mL concentration of polymeric nanoparticles in an aqueous medium; wherein the polymeric nanoparticles comprise: a poly(lactic) acid-block-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(gly colic) acid-block-poly(ethylene)glycol copolymer, and a taxane agent; 4 to 6 weight percent sucrose or trehalose; and 7 to 12 weight percent hydroxypropyl ⁇ -cyclodextrin, as disclosed in US 8637083 to Troiano et al. (BIND
  • the conjugates and/or particles of the invention may be delivered with a bacteriophage.
  • a bacteriophage may be conjugated through a labile/non labile linker or directly to at least 1 ,000 therapeutic drug molecules such that the drug molecules are conjugated to the outer surface of the bacteriophage as disclosed in US 201 10286971 to Yacoby et al, the content of which is incorporated herein by reference in its entirety.
  • the bacteriophage may comprise an exogenous targeting moiety that binds a cell surface molecule on a target cell.
  • the conjugates and/or particles of the invention may be delivered with a dendrimer.
  • the conjugates may be encapsulated in a dendrimer, or disposed on the surface of a dendrimer.
  • the conjugates may bind to a scaffold for dendritic encapsulation, wherein the scaffold is covalently or non-covalently attached to a polysaccharide, as disclosed in US 20090036553 to Piccariello et al., the content of which is incorporated herein by reference in its entirety.
  • the scaffold may be any peptide or oligonucleotide scaffold disclosed by Piccariello et al.
  • the conjugates and/or particles of the invention may be delivered by a cyclodextrin.
  • the conjugates may be formulated with a polymer comprising a cyclodextrin moiety and a linker moiety as disclosed in US
  • the conjugates and/or particles of the invention may be delivered with an aliphatic polymer.
  • the aliphatic polymer may comprise polyesters with grafted zwitterions, such as polyester-graft-phosphorylcholine polymers prepared by ring-opening polymerization and click chemistry as disclosed in US 8802738 to Emrick; the content of which is incorporated herein by reference in its entirety.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's The Science and Practice of Pharmacy 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl- pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked poly(vinyl- pyrrolidone)
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol
  • carbomers e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer
  • carrageenan cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxy ethylene sorbitan monolaurate
  • TWEEN®20 polyoxy ethylene sorbitan [TWEENn®60], polyoxy ethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether
  • binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
  • natural and synthetic gums e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose
  • polymethacrylates are polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabi sulfite, GLYDANT PLUS®, PHENONIP®, methylparaben,
  • GERMALL® 115 GERMABEN®II, NEOLONETM, KATHONTM, and/or EUXYL®.
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl my ristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • B. Lipidoids such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • Lipidoids may be used to deliver conjugates of the present invention.
  • Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the conjugates of the present invention, for a variety of therapeutic indications including vaccine adjuvants, following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
  • Lipidoid complexes of conjugates of the present invention can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to conjugates of the present invention.
  • lipidoid formulations for the localized delivery of conjugates to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the conjugates.
  • the conjugates of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of the conjugates of the invention include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to- batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-NN- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety).
  • DODMA 1,2-dioleyloxy-NN- dimethylaminopropane
  • DLin-DMA 1,2-dioleyloxy-3-dimethylaminopropane
  • DLin-KC2-DMA 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dio
  • the conjugates of the invention may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
  • the conjugates of the invention may be formulated in a lipid- poly cation complex.
  • the formation of the lipid-poly cation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety.
  • the poly cation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326; herein incorporated by reference in its entirety.
  • a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326; herein incorporated by reference in its entirety.
  • the conjugates of the invention may be formulated in a lipid-poly cation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the
  • the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No.
  • the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of which is herein incorporated by reference in their entirety.
  • the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No.
  • the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; each of which is herein incorporated by reference in their entirety.
  • the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is herein incorporated by reference in their entirety.
  • conjugates described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or WO2008103276; each of which is herein incorporated by reference in their entirety.
  • conjugates described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No. 20120207845; herein incorporated by reference in its entirety.
  • the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a conjugate.
  • the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121 ; herein incorporated by reference in its entirety).
  • Nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial membranes
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photo bleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photo bleaching
  • MPT high resolution multiple particle tracking
  • compositions which can penetrate a mucosal barrier may be made as described in U. S. Pat. No. 8,241 ,670, herein incorporated by reference in its entirety.
  • Nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
  • the polymeric material may include, but is not limited to, polyamines, poly ethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,
  • polystyrene resin polystyrene resin
  • polystyrene resin polystyrene resin
  • the polymeric material may be biodegradable and/or biocompatible.
  • the polymeric material may additionally be irradiated.
  • the polymeric material may be gamma irradiated (See e.g., International App. No. WO201282165, herein incorporated by reference in its entirety).
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(gly colic acid) (PGA), poly(lactic acid-co-gly colic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (
  • the nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety).
  • the co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
  • the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the conjugate of the invention is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of therapeutic agents (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of therapeutic agents (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg
  • such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells (e.g., antigen presenting cells, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells and leukocytes), tumor cells and endothelial cells, (Akinc et al. Mol Ther. 2010 18: 1357-1364; Song et al, Nat Biotechnol. 2005 23 :709-717; Judge et al., J Clin Invest.
  • immune cells e.g., antigen presenting cells, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells and leukocytes
  • tumor cells and endothelial cells e.g., endothelial cells
  • Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al, Crit Rev Ther Drug Carrier Syst. 2008 25: 1 -61 ; Benoit et al., Biomacromolecules .
  • the conjugates of the invention are formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm.
  • SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al, ACSNano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).
  • the conjugates of the invention can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattem of release to effect a therapeutic outcome.
  • the conjugates of the invention may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the conjugates of the invention, encapsulation may be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of conjugate of the invention may be enclosed, surrounded or encased within the particle.
  • Partially encapsulation means that less than 10, 10, 20, 30, 40 50 or less of the conjugate of the invention may be enclosed, surrounded or encased within the particle. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the particle.
  • the conjugates of the invention may be encapsulated into a nanoparticle or a rapidly eliminated nanoparticle and the nanoparticles or a rapidly eliminated nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
  • the nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the conjugate formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxy ethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as
  • ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the conjugate of the present invention may be encapsulated in a therapeutic nanoparticle.
  • Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos.
  • WO2010005740 WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285,
  • therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.
  • the therapeutic nanoparticle may be formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the conjugate of the present invention (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticles may be formulated to be target specific.
  • the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518 herein incorporated by reference in its entirety).
  • the therapeutic nanoparticles of the present invention may be formulated to be antiviral immunotherapeutics or vaccine adjuvants.
  • the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in their entirety.
  • the nanoparticles of the present invention may comprise a polymeric matrix.
  • the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
  • polyurethanes polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the therapeutic nanoparticle comprises a diblock copolymer.
  • the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester) or combinations thereof.
  • a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumer
  • the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).
  • the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910; each of which is herein incorporated by reference in its entirety).
  • the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g.,
  • the therapeutic nanoparticle may comprise at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxy ethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
  • the therapeutic nanoparticles may comprise at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,
  • poly(amidoamine) dendrimers poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference in its entirety) and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one degradable polyester which may contain poly cationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the therapeutic nanoparticle may include a conjugation of at least one targeting ligand.
  • the targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its entirety).
  • the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No. WO2011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).
  • the conjugates of the invention may be encapsulated in, linked to and/or associated with synthetic nanocarriers.
  • Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411 and WO2012149454 and US Pub. Nos.
  • the synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. WO2010005740,
  • the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. WO2011072218 and US Pat No. 8,211,473; each of which is herein incorporated by reference in their entirety.
  • the synthetic nanocarriers may contain reactive groups to release the conjugates described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety).
  • the synthetic nanocarriers may be formulated for targeted release.
  • the synthetic nanocarrier is formulated to release the conjugates at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle may be formulated to release the conjugates after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entirety).
  • the synthetic nanocarriers may be formulated for controlled and/or sustained release of conjugates described herein.
  • the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.
  • the nanoparticle may be optimized for oral administration.
  • the nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof.
  • the nanoparticle may be formulated by the methods described in U.S. Pub. No. 20120282343; herein incorporated by reference in its entirety.
  • the conjugates of the invention can be formulated using natural and/or synthetic polymers.
  • Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYC ON JUGATE® (Arrowhead Research Corp.,
  • PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-gly colic acid) (PLGA) polymers,
  • RONDELTM RNAi/Oligonucleotide Nanoparticle Delivery
  • PHASERXTM pH responsive co-block polymers
  • a non-limiting example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No.
  • Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.
  • the polymers used in the present invention have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer.
  • the polymer may be processed by methods known and/or described in the art and/or described in International Pub. No.
  • a non-limiting example of PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N- methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • PLGA injectable depots e.g., ELIGARD® which is formed by dissolving PLGA in 66% N- methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • the pharmaceutical compositions may be sustained release formulations.
  • the sustained release formulations may be for subcutaneous delivery.
  • Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®
  • modified mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the conjugate in the PLGA microspheres while maintaining the integrity of the conjugate during the encapsulation process.
  • EVAc are non-biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters).
  • Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene- poly oxy ethylene having a low viscosity at temperatures less than 5°C and forms a solid gel at temperatures greater than 15°C.
  • PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days.
  • GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
  • Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et al, Biomacromolecules. 201 1 12:2708-2714; Rozema et al, Proc Natl Acad Sci U S A. 2007 104: 12982-12887; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature, 2010, 464: 1067-1070; each of which is herein incorporated by reference in its entirety).
  • GalNAc N- acetylgalactosamine
  • the conjugates of the invention may be formulated with or in a polymeric compound.
  • the polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethylenimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-gly colic acid) (PAGA), biodegradable cross
  • the conjugates of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its entirety.
  • the conjugate may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.
  • the conjugate of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which are herein incorporated by reference in their entireties) or PLGA- PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, herein incorporated by reference in its entirety).
  • the conjugate of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).
  • a poly amine derivative may be used to deliver conjugates of the invention or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817 herein incorporated by reference in its entirety).
  • a pharmaceutical composition may include the conjugates of the invention and the polyamine derivative described in U.S. Pub. No. 20100260817 (the contents of which are incorporated herein by reference in its entirety).
  • the conjugates of the invention may be delivered using a polyamide polymer such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; herein incorporated by reference in its entirety).
  • a polyamide polymer such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; herein incorporated by reference in its entirety).
  • the conjugate of the invention may be formulated with at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxy ethyl
  • the conjugates of the invention may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos.
  • the conjugates of the invention may be formulated with a polymer of formula Z as described in WO2011115862, herein incorporated by reference in its entirety.
  • the conjugates of the invention may be formulated with a polymer of formula Z, Z' or Z" as described in International Pub. Nos. WO2012082574 or
  • WO2012068187 each of which are herein incorporated by reference in their entireties.
  • the polymers formulated with the conjugates of the present invention may be synthesized by the methods described in International Pub. Nos. WO2012082574 or WO2012068187, each of which are herein incorporated by reference in their entireties.
  • Formulations of conjugates of the invention may include at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,
  • the conjugate of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof.
  • the biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Pat. No.
  • the poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety.
  • the biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos.
  • the linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886.
  • the PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912 herein incorporated by reference in its entirety.
  • the PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyarginine, polyomithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides).
  • the biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U. S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are herein incorporated by reference in their entireties.
  • the multi- block copolymers may be synthesized using linear polyethylenimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
  • LPEI linear polyethylenimine
  • the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U. S. Pub. No. 20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.
  • the conjugates of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the conjugate of the invention may be formulated with at least one cross linkable polyester.
  • Cross linkable polyesters include those known in the art and described in US Pub. No. 20120269761, herein incorporated by reference in its entirety.
  • the polymers described herein may be conjugated to a lipid- terminating PEG.
  • PLGA may be conjugated to a lipid- terminating PEG forming PLGA-DSPE-PEG.
  • PEG conjugates for use with the present invention are described in International Publication No. WO2008103276, herein incorporated by reference in its entirety.
  • the polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U. S. Pat. No. 8,273,363, herein incorporated by reference in its entirety.
  • the conjugates of the invention may be conjugated with another compound.
  • conjugates are described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
  • the conjugates of the invention may be conjugated with conjugates of formula 1-122 as described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
  • the modified RNA described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc.
  • the conjugates of the invention may be conjugated and/or encapsulated in gold-nanoparticles.
  • the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups.
  • the polymer formulation may be contacted with a cationic lipopolymer using the methods described in U. S. Pub. No. 20090042829 herein incorporated by reference in its entirety.
  • the cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside- polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l ,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), N-[l- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3- dioleyl
  • DOSPA trifluoroacetate
  • DC-Cholesterol HC1 diheptadecylamidoglycyl spermidine
  • DOGS N,N- distearyl-N,N-dimethylammonium bromide
  • DDAB N,N- distearyl-N,N-dimethylammonium bromide
  • DMRIE N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide
  • DODAC trifluoroacetate
  • the conjugates of the invention may be formulated in a polyplex of one or more polymers (U. S. Pub. No. 20120237565 and 20120270927; each of which is herein incorporated by reference in its entirety).
  • the polyplex comprises two or more cationic polymers.
  • the catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
  • the conjugates of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
  • Components may be combined in a core-shell, hybrid, and/or layer- by -layer architecture, to allow for fine-tuning of the nanoparticle so that delivery of the conjugates of the invention may be enhanced (Wang et al., Nat Mater. 2006, 5:791 -796; Fuller et al, Biomaterials. 2008, 29: 1526-1532; DeKoker et al, Adv Drug Deliv Rev.
  • the nanoparticle may comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (International Pub. No. WO20120225129; herein incorporated by reference in its entirety).
  • hydrophilic-hydrophobic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers International Pub. No. WO20120225129
  • Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver therapeutic agents in vivo.
  • a lipid coated calcium phosphate nanoparticle which may also contain a targeting ligand such as anisamide, may be used to deliver the conjugate of the present invention.
  • a targeting ligand such as anisamide
  • a lipid coated calcium phosphate nanoparticle was used (Li et al, J Contr Pel. 2010, 142: 416-421 ; Li et al, J Contr Rel. 2012, 158: 108-114; Yang et al, Mol Ther. 2012, 20:609-615; herein
  • This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the therapeutic agent.
  • a PEG-charge-conversional polymer (Pitella et al,
  • Biomaterials. 201 1, 32:3106-31 14 may be used to form a nanoparticle to deliver the conjugate of the present invention.
  • the PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a poly cation at acidic pH, thus enhancing endosomal escape.
  • core-shell nanoparticles have additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al, Proc Natl Acad Sci USA. 201 1, 108: 12996-13001).
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • the core-shell nanoparticles may efficiently deliver a therapeutic agent to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
  • core-shell nanoparticles have additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al, Proc Natl Acad Sci USA. 2011 , 108: 12996-13001).
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • the core-shell nanoparticles may efficiently deliver a therapeutic agent to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
  • the lipid nanoparticles may comprise a core of the conjugates disclosed herein and a polymer shell.
  • the polymer shell may be any of the polymers described herein and are known in the art.
  • the polymer shell may be used to protect the modified nucleic acids in the core.
  • Core-shell nanoparticles for use with the conjugates of the present invention are described and may be formed by the methods described in U. S. Pat. No. 8,313,777 herein incorporated by reference in its entirety.
  • the core-shell nanoparticles may comprise a core of the conjugates disclosed herein and a polymer shell.
  • the polymer shell may be any of the polymers described herein and are known in the art.
  • the polymer shell may be used to protect the modified nucleic acid molecules in the core.
  • Inorganic nanoparticles exhibit a combination of physical, chemical, optical and electronic properties and provide a highly multifunctional platform to image and diagnose diseases, to selectively deliver therapeutic agens, and to sensitive cells and tissues to treatment regiments.
  • enhanced permeability and retention (EPR) effect provides a basis for the selective accumulation of many high- molecular-weight drugs.
  • Circulating inorganic nanoparticles preferentially accumulate at tumor sites and in inflamed tissues (Yuan et al., Cancer Res., vol.55(17): 3752-6, 1995, the contents of which are incorporated herein by reference in their entirety) and remain lodged due to their low diffusivity (Pluen et al., PNAS, vol.98(8):4628-4633, 2001 , the contents of which are incorporated herein by reference in their entirety).
  • the size of the inorganic nanoparticles may be 10 nm - 500 nm, 10 nm - 100 nm or 100 nm - 500 nm.
  • the inorganic nanoparticles may comprise metal (gold, iron, silver, copper, nickel, etc.), oxides (ZnO, TiC , AkCb, SiC , iron oxide, copper oxide, nickel oxide, etc.), or semiconductor (CdS, CdSe, etc.).
  • the inorganic nanoparticles may also be perfluorocarbon or FeCo.
  • Inorganic nanoparticles have high surface area per unit volume. Therefore, they may be loaded with therapeutic drugs and imaging agents at high densitives.
  • a variety of methods may be used to load therapeutic drugs into/onto the inorganic nanoparticles, including but not limited to, colvalent bonds, electrostatic interactions, entrapment, and encapsulation.
  • the inorganic nanoparticles may be funcationalized with targeting moieties, such as tumor-targeting ligands, on the surface. Formulating therapeutic agents with inorganic nanoparticles allows imaging, detection and monitoring of the therapeutic agents.
  • the conjugate of the invention is hydrophobic and may be form a kinetically stable complex with gold nanoparticles funcationalized with water-soluble zwitterionic ligands disclosed by Kim et al. (Kim et al, JACS, vol. l31(4): 1360-1361 , 2009, the contents of which are incorporated herein by reference in their entirety). Kim et al.
  • the conjugates of the invention may be formulated with gold nanoshells.
  • the conjugates may be delivered with a temperature sensitive system comprising polymers and gold nanoshells and may be released
  • Sershen et al. designed a delivery vehicle comprising hydrogel and gold nanoshells, wherein the hydrogels are made of copolymers of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) and the gold nanoshells are made of gold and gold sulfide (Sershen et al, J Biomed Mater, vol.51 :293-8, 2000, the contents of which are incorporated herein by reference in their entirety). Irradiation at 1064 nm was absorbed by the nanoshells and converted to heat, which led to the collapse of the hydrogen and release of the drug.
  • the conjugate of the invention may also be encapsulated inside hollow gold nanoshells.
  • the conjugates of the invention may be attached to gold nanoparticles via covalent bonds. Covalent attachment to gold nanoparticles may be achieved through a linker, such as a free thiol, amine or carboxylate functional group.
  • the linkers are located on the surface of the gold nanoparticles.
  • the conjugates of the invention may be modified to comprise the linkers.
  • the linkers may comprise a PEG or oligoethylene glycol moiety with varying length to increase the particles' stability in biological environment and to control the density of the drug loads. PEG or oligoethylene glycol moieties also minimize nonspecific adsorption of undesired biomolecules.
  • PEG or oligoethylene gycol moieties may be branched or linear.
  • Tong et al. disclosed that branched PEG moieties on the surface of gold nanoparticles increase circulatory half-life of the gold nanoparticles and reduced serum protein binding (Tong et al, Langmuir, vol.25(21): 12454-9, 2009, the contents of which are incorporated herein by reference in their entirety).
  • the conjugate of the invention may comprise PEG-thiol groups and may attach to gold nanoparticles via the thiol group.
  • the synthesis of thiol-PEGylated conjugates and the attachment to gold nanoparticles may follow the method disclosed by El- Say ed et al. (El-Sayed et al, Bioconjug. Chem., vol.20(12):2247-2253, 2010, the contents of which are incorporated herein by reference in their entirety).
  • the conjugate of the invention may be tethered to an amine- functionalized gold nanoparticles.
  • Lippard et al. disclosed that Pt(IV) prodrugs may be delivered with amine-functionalized polyvalent oligonucleotide gold nanoparticles and are only activated into their active Pt(II) forms after crossing the cell membrane and undergoing intracellular reduction (Lippard et al, JACS, vol.131 (41): 14652-14653, 2009, the contents of which are incorporated herein by reference in their entirety).
  • the cytotoxic effects for the Pt(IV)-gold nanoparticle complex are higher than the free Pt(IV) drugs and free cisplatin.
  • conjugates of the invention are formulated with magnetic nanoparticle such as iron, cobalt, nickel and oxides thereof, or iron hydroxide nanoparticles.
  • Localized magnetic field gradients may be used to attract magnetic nanoparticles to a chosen site, to hold them until the therapy is complete, and then to remove them.
  • Magnetic nanoparticles may also be heated by magnetic fields.
  • Alexiou et al. prepared an injection of magnetic particle, Ferro fluids (FFs), bound to anticancer agents and then concentrated the particles in the desired tumor area by an external magnetic field (Alexiou et al., Cancer Res. vol.60(23):6641 -6648, 2000, the contents of which are incorporated herein by reference in their entirety). The desorption of the anticancer agent took place within 60 min to make sure that the drug can act freely once localized to the tumor by the magnetic field.
  • FFs Ferro fluids
  • the conjugates of the invention are loaded onto iron oxide nanoparticles.
  • the conjugates of the invention are formulated with super paramagnetic nanoparticles based on a core consisting of iron oxides (SPION).
  • SPION super paramagnetic nanoparticles based on a core consisting of iron oxides (SPION).
  • SPION are coated with inorganic materials (silica, gold, etc.) or organic materials (phospholipids, fatty acids, polysaccharides, peptides or other surfactants and polymers) and can be further functionalized with drugs, proteins or plasmids.
  • water-dispersible oleic acid (OA)-poloxamer-coated iron oxide magnetic nanoparticles disclosed by Jain et al. (Jain, Mol. Pharm., vol.2(3): 194-205, 2005, the contents of which are incorporated herein by reference in their entirety) may be used to deliver the conjugates of the invention.
  • Therapeutic drugs partition into the OA shell surrounding the iron oxide nanoparticles and the poloxamer copolymers (i.e., Pluronics) confers aqueous dispersity to the formulation.
  • Pluronics poloxamer copolymers
  • the conjugates of the invention are bonded to magnetic nanoparticles with a linker.
  • the linker may be a linker capable of undergoing an
  • the conjugates of the invention may be delivered with a drug delivery system disclosed in US 7329638 to Yang et al, the contents of which are incorporated herein by reference in their entirety.
  • the drug delivery system comprises a magnetic nanoparticle associated with a positively charged cationic molecule, at least one therapeutic agent and a molecular recognition element.
  • nanoparticles having a phosphate moiety are used to deliver the conjugates of the invention.
  • the phosphate-containing nanoparticle disclosed in US 8828975 to Hwu et al, the contents of which are incorporated herein by reference in their entirety, may be used.
  • the nanoparticles may comprise gold, iron oxide, titanium dioxide, zinc oxide, tin dioxide, copper, aluminum, cadmium selenide, silicon dioxide or diamond.
  • the nanoparticles may contain a PEG moiety on the surface.
  • the conjugate of the invention can be formulated with peptides and/or proteins in order to increase peneration of cells by the conjugates of the invention.
  • peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations.
  • a non- limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention include a cell-penetrating peptide sequence attached to poly cations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al, Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al, Curr. Pharm. Des. 2003,
  • compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space.
  • a cell penetrating agent e.g., liposomes
  • the conjugates of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologies (Cambridge, MA) in order to enable intracellular delivery (Cronican et & ⁇ ., ACS Chem. Biol.
  • the cell-penetrating polypeptide may comprise a first domain and a second domain.
  • the first domain may comprise a supercharged polypeptide.
  • the second domain may comprise a protein-binding partner.
  • protein-binding partner includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides.
  • the cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner.
  • the cell-penetrating polypeptide may be capable of being secreted from a cell where conjugates of the invention may be introduced.
  • compositions of the present invention may be formulated as vaccines, such as cancer vaccines.
  • Cancer vaccines aim to augment immune responses with the tumor antigen-expressing targets already present, such as by inducing antigen specific T cells.
  • the general composition of cancer vaccines may include a source of TAAs and adjuvants that results in activation of dendritic cells for productive antigen presentation.
  • the adjuvants may be oil-based formulations, Toll like receptor (TLR) ligands, recombinant cytokines or the natural innate ligands.
  • TLR Toll like receptor
  • Adjuvants may be aluminium based adjuvants including but not limiting to aluminium hydroxide and aluminium phosphate; saponins such as steroid saponins and triterpenold saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines and routes of administrations.
  • Adjuvants may include, but are not limited to, alpha glucose bearing
  • glycosphingolipid compounds disclosed by Chen et al (US Patent publication NO. 2015/0071960, the content of which is incorporated herein by reference in its entirety).
  • Those compounds when added into the present particles in combination with conjugates of the present invention can elevate invariant natural killer T (iNKT) cells and increases cytokine and/or chemokine production, where the cytokine production is sufficient to transactivate downstream immune cells including dendritic cells, natural killer cells, B cells, CD+4 T and CD8+ T cells.
  • iNKT invariant natural killer T
  • adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation.
  • Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs).
  • APCs antigen presenting cells
  • DCs dendritic cells
  • Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity.
  • adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation.
  • This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures (Powell et al, Clin Exp. Vaccine Res. , Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. 2015, 4(l):23-45).
  • heat shock proteins or their peptide derivatives may be used as an adjuvant in the composition as disclosed in Shevtsov et al. ⁇ Frontiers in Immunology, vol.7:article 171 (2016)).
  • HSP70 protein, HSP70 peptide derived thereof, HSP90 protein, HSP90 peptide derived thereof may be combined with conjuates or particles of the present invention to produce vaccine compositions.
  • the vaccine composition comprises conjugates or particles of the present invention and synthetic toll like receptor-4 (TLR-4) agonist peptides disclosed in Shanmugam et al. (PloS ONE, vol.7(2):e30839 (2012)) as adjuvants.
  • TLR-4 synthetic toll like receptor-4
  • the vaccine composition comprises conjugates or particles of the present invention and pidotimod as an adjuvant.
  • conjugates of the present invention may be formulated as peptide based vaccines. Such vaccines may target directly to dendritic cells in vivo to activate dendritic cells for presenting antigens.
  • Conjugates may be formulated as micro- or nanoparticles, liposomes and/or virus-like particles (VLP) to increase intracellular membrane permeability.
  • VLP virus-like particles
  • nanoparticle cancer vaccines may be formulated to deliver several TAAs and adjuvants simultaneously, enabling a coordinated activation of
  • nanoparticles can also be functionalized in order to actively target dendritic cells in vivo, to increase their cellular internalization and
  • nanoparticle formulations as described may be modified for imaging, diagnostic, and targeted delivery of conjugates to immune cells.
  • the conjugate of the present invention may be delivered with a liposomal drug delivery system as reported by van Broekhoven, CL., et al, Cancer Res. 2004, 12:4357-65; the contents of which are incorporated herein by reference in their entirety, which targets dendritic cells as a platform to induce a highly effective immunity against tumor cells.
  • liposome-DNA complexes have also been described, constituting an effective strategy to elicit anti-tumor immunity (U'Ren, L., et al, Cancer Gene Ther. 2006, 1 1 : 1033-44; the contents of which are incorporated herein by reference in their entirety).
  • the conjugate of the present invention may be formulated into self-assembling spherical polymeric micelles formed by amphiphilic block copolymers in an aqueous medium.
  • a hydrophobic core and a hydrophilic surface compose these structures and their size ranges from 10 to 100 nm (Torchilin, VP., J. Control. Release 2001 , 73(2- 3): 137-72; the contents of which are incorporated herein by reference in their entirety).
  • novel pH-responsive polymer micelles formed by an N-(2- hydroxypropyl) methacrylamide corona and a propylacrylic acid (PAA)/dimethylaminoethyl methacrylate (DMAEMA)/butyl methacrylate (BMA) core have been investigated for antigen trafficking modulation in dendritic cells.
  • PAA propylacrylic acid
  • DMAEMA dimethylaminoethyl methacrylate
  • BMA butyl methacrylate
  • micelles formed by DMAEMA and pyridyl disulfide ethyl methacrylate (PDSEMA), carrying both short single-stranded synthetic DNA molecules which contain a cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide (CpG ODN) and protein antigens have shown to elicit and increase the cellular and humoral immune response by modulating and stimulating antigen cross-presentation, as summarized by Wilson JT., et al, A CS Nano. 2013, 7(5):3912-25; the contents of which are incorporated herein by reference in their entirety.
  • polymers from different origins already described as useful materials for polymeric nanoparticle production and used in other preclinical studies may be formulated with the conjugate of the present invention.
  • Polymers can be from natural origin, such as chitosan, or synthesized, as polylactic acid and poly-lactic-co-gly colic acid (PLGA).
  • PCL has the characteristics of an antigen controlled release matrices by its low degradation rate, hydrophobicity, good drug permeability, in vitro stability and low toxicity.
  • the adjuvant effect of PCL nanoparticles to induce immune responses against an infectious disease was previously confirmed by several studies (Benoit, MA., et al. j/wi. J. Pharm. 1999, 184(1):73- 84; Florindo, HF., et al, Vaccine. 2008, 26(33):4168-77; Florindo, HF., et al., Biomaterials.
  • these nanoparticles may be modified with maturation signals at their surface for direct ligand-receptor interaction, as mannose receptors are overexpressed at dendritic cell and macrophage cell surfaces.
  • the conjugate of the present invention may be loaded into PLGA nanoparticles with melanoma antigens to elicit effective anti-tumor activity by CTLs in vivo (Zhang, Z., et al, Biomaterials. 2011 , 32(14):3666-78; Ma, W., et al, Int. J. Nanomedicine. 2012, 7: 1475-87; the contents of each of which are incorporated herein by reference in their entirety).
  • chitosan nanoparticles targeting dendritic cells carrying IL-12 were administered in an animal model that resulted in suppression of tumor growth and increased induction of apoptosis (Kim, TH., et al, Mol Cancer Ther. 2006, 5(7): 1723-32; the contents of which are incorporated herein by reference in their entirety).
  • the conjugate of the present invention may be delivered with a hyper-branched spherical dendrimer nanocarrier with a hydrophilic surface and a hydrophobic central core.
  • the linear poly(glutamic acid) is a poly(amino acid) polymer has been reported to have considerable potential for antigen delivery to dendritic cells, adjuvant properties for dendritic cell maturation, and able to induce CTLs (Yoshikawa, T., et al., Vaccine. 2008, 26(10): 1303-13; the contents of which are incorporated herein by reference in their entirety).
  • compositions and formulations containing an effective amount of conjugates or particles of the present invention may be administered to a subject in need thereof by any route which results in a therapeutically effective outcome in said subject.
  • routes include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal),
  • intraperitoneal intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal,
  • compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • particles, nanoparticles and/or polymeric nanoparticles are administered to bone marrow.
  • particles, nanoparticles and/or polymeric nanoparticles are administered to areas having a lot of dendritic cells, such as subcutaneous space.
  • compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • split dosing regimens such as those described herein may be used.
  • a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a “total daily dose” is an amount given or prescribed in 24 hr. period. It may be administered as a single unit dose.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous.
  • the dosage forms may be liquid dosage forms.
  • Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
  • compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • the dosages forms may be injectable.
  • Inj ectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents include, but are not limited to, water, Ringer's solution, U. S. P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Injectable depot forms are made by forming microencapsule matrices of the conjugates in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of conjugates to polymer and the nature of the particular polymer employed, the rate of active agents in the conjugates can be controlled.
  • biodegradable polymers examples include, but are not limited to, poly(orthoesters) and poly (anhydrides). Depot injectable formulations may be prepared by entrapping the conjugates in liposomes or microemulsions which are compatible with body tissues.
  • solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • compositions of the present invention may be used to hamess the immune system to eliminate tumor cells.
  • compositions of the present invention may be used as vaccines.
  • Vaccines may be peptide vaccines such as conjugates, particles comprising conjugates of TAAs, TAA epitopes or derivatives thereof; or dendritic cell vaccines to increase the frequency of tumor specific cytotoxic T lymphocytes, wherein the DCs are primed with conjugates and/or particles comprising conjugates of TAAs, TAA epitopes or derivatives thereof; or adoptive cellular immunotherapies involving adoptive transfer of effector T cells which are actived by compositions of the present inventions.
  • compositions as discussed above may be used either for active immunotherapy and adoptive immunotherapy to prevent/treat a disease such as cancer.
  • compositions of the present invention may be used to prime and amplify Tumor antigen specific T cells in vivo.
  • T cells are isolated from a subject to be treated and may be primed and amplified using compositions of the present invention ex vivo prior to their infusion.
  • Adoptive immunotherapy is a procedure whereby an individual's own T cells are expanded ex vivo and re-infused back into the body.
  • particles of the present invention may facilitate the delivery of the T cells that have been activated ex vivo. Both active and adoptive immunotherapy can be used as therapeutic strategies for treatment of cancer.
  • compositions of the present invention may be used for antibody-based cancer immunotherapy.
  • Conjugates comprising antibodies or derivatives thereof may be used for this therapy.
  • compositions of the present invention may be used for cytokine based cancer immunotherapy.
  • conjugates comprising one or more tumor antigenic peptides, and/or particles and formulations comprising such conjugates may be used as peptide vaccines to treat a tumor.
  • Peptide vaccines may be used to induce antibodies that can react with the native protein expressed in tumor cells and promote complement-mediated lysis of tumor cells, or elicit T cell based cellular immune response to destroy tumor cells, or both.
  • tumor antigens of interest to cancer vaccine development have been categorized into "unique or neoantigens,” which are unique to a tumor tissue and are not present in normal tissue and "shared antigens" which are common among two or more tumors or populations.
  • Shared antigens can fall into several categories.
  • the first category is composed of certain shared tumor associated antigens derived from proteins that are expressed in cancer but not expressed in most normal tissues.
  • This category includes the cancer/ testis (CT) antigens, which are expressed in certain tumors, but in normal tissue are found only in placental trophoblasts and testicular germ cells.
  • CT cancer/ testis
  • a tumor antigen may be an antigen that is specific to the tissue in which the tumor arises. Because of their limited tissue distribution, the CT antigens or lineage/tissue specific antigens may not cause "off target" immune response-related toxicities, even if high-affinity T cells are elicited upon antigen vaccination.
  • the third group under consideration is consists of certain tumor associated antigens, which arise from genes which are overexpressed in certain tumors, but are also expressed in normal tissues, albeit at lower levels. This group is more precarious, since an immunotherapeutic approach targeting these antigens may result in side effects in the normal tissues if a certain threshold of response is overstepped.
  • mice and humans can mount T-cell responses against neoantigens, and mice are tumor protected by immunization with a single mutated peptide.
  • memory cytotoxic T lymphocyte (CTL) responses to neoantigens are generated in patients with unexpected long-term survival or those who have undergone effective immunotherapy.
  • immunotherapeutically useful progenitor sequences comprising or encoding neoepitopes (antigens or immunogens).
  • the first type are somatic point mutations, which lead to the expression of one or more different amino acids in the protein in the tumor.
  • Other mutations lead to the generation of entirely novel tumor-specific protein sequences (progenitor sequences).
  • These include frameshift mutations, which can be either insertions or deletions, and which lead to a new open reading frame with a novel tumor-specific protein sequence.
  • Read-through mutations in which a stop codon is modified or deleted, allow the translation of a longer protein, and thereby also generate a novel tumor specific progenitor protein sequence.
  • Splice site mutations cause the inclusion of an intron into the mature mRNA and thus a unique tumor-specific progenitor protein sequence.
  • any of the foregoing types of antigens or neoantigen peptides identified on neoORFs and missense neoantigens are synthesized in vitro and linked to conjugates of the present invention.
  • the conjugates and compositions thereof may be administered to a subject with a powerful immune adjuvant and coupled with complementary immunotherapeutics such as checkpoint-blockade inhibitors.
  • Antigen presenting cells in particular the professional APCs: dendritic cells (DCs) function at the frontier of the immune system and at the interface of the innate and adaptive immune responses, making them uniquely suited for cancer immunotherapy.
  • APC antigen presenting cells
  • DCs dendritic cells
  • conjugates, and particles and/or formulations packaging conjugates may be used to harness dendritic cells (DCs) to enhance antigen presentations.
  • conjugates comprising tumor antigens as payloads may be used to prime dendritic cells and primed DCs then can be used as cellular vaccines for treating a cancer.
  • ex vivo DCs are generated from in vitro differentiation of peripheral blood mononuclear cells (PBMCs) in the presence of stimulating factors including granulocyte-macrophage colony- stimulating factor (GM-CSF) and interleukin (IL)-4 or IL-13 (Alters SE et al, IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy, J Immunother., 1999, 22: 229-236).
  • stimulating factors including granulocyte-macrophage colony- stimulating factor (GM-CSF) and interleukin (IL)-4 or IL-13
  • the DCs can comprise TAA peptides of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more conjugates comprising one or more antigenic peptides. As a non-limiting example, to induce a strong and durable anti-tumor T cell responses, conjugates comprising multiple TAA peptide epitopes may be used to pulse DCs. In general, in vitro cultured autologous DCs are transformed with conjugates comprising one or more TAA peptide epitopes. Pulsed DCs may be amplifies in vitro before infusion.
  • DC cellular vaccines are dendritic cells that comprise one or more antigenic peptides included in the present compositions.
  • Adoptive T cell transfer is a direct strategy to increase the frequency of tumor antigen specific T cells.
  • Tumor antigen specific T cells can be largely expanded in vitro, thus by -pass the early stages of endogenous T cell activation.
  • conjugates comprising TAA peptide epitopes, alone or in combination with so-stimulatory agents may be coupled to the surface of APCs (e.g., DCs) to activate T cells in vitro.
  • APCs e.g., DCs
  • conjugates, particles and formulations comprising conjugates may be used to enhance T cells mediated immune response, particularly anti-cancer immune response.
  • the T cells may be from a variety of sources such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a subject. If obtained from a subject, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues (e.g., tumor tissue) or fluids. T cells can also be enriched for or purified.
  • T cell is a human T cell .
  • the T cell can be any type of T lymphocyte and can be of any developmental stage, including but not limited to, CD4 + /CD8 + double positive T lymphocytes, CD4 + helper T lymphocytes, e.g., Thi and Th2 cells, CD8 + T lymphocytes (e.g., cytotoxic T lymphocytes), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, and the like.
  • CD4 + /CD8 + double positive T lymphocytes CD4 + helper T lymphocytes, e.g., Thi and Th2 cells
  • CD8 + T lymphocytes e.g., cytotoxic T lymphocytes
  • PBMCs peripheral blood mononuclear cells
  • PBLs peripheral blood leukocytes
  • TILs tumor infiltrating lymphocytes
  • memory T cells naive T
  • TIL tumor infiltrating lymphocytes
  • TILs tumor infiltrating lymphocytes
  • CTLs CD8+ cytotoxic T lymphocytes
  • conjugates comprising co-stimulatory molecules, and/or cytokines that can support T cell growth and maintenance, may be used to expand in vitro TLRs.
  • a patient, prior to T cell infusion is conditioned with a lymphodepleting regimen, and then is given IL-2 post infusion.
  • CD4+ or CD8+ antigen specific T cell clones Some studies have proven that neoantigens of mutated peptides identified in a cancer subject may be the major natural targets of tumor specific TILs (Lenneiz et al, The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci USA, 2005, 102: 16013- 16018). Accordingly, conjugates comprising tumor specific -neoantigens may be used ex vivo for expanding patient derived T cells such as PBMCs before adoptive T cell therapy.
  • conjugates comprising tumor specific neoantigens may further comprises one or more non-specific T cell receptor stimulating agent as payloads, wherein the non-specific T cell receptors stimulators may be a T cell growth factor, including but not limited to interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.
  • IL-12 is a preferred T-cell growth factor.
  • the T cell growth factor may be included in the same conjugate as one or more tumor specific antigens, or the T cell growth factor may be in separate conjugate but is packaged together with the conjugates comprising one or more tumor specific antigens in the same particle or other formulations.
  • the method for producing activated cytotoxic T lymphocytes comprises contacting in vitro autologous T cells from a patient himself/herself with TAA antigenic peptide loaded class I MHC/HLA molecules expressed on the surface of a suitable APC (e.g., a DC) or an artificial composition mimicking an antigen-presenting cell for a period of time sufficient to activate said CTL in an antigen specific manner.
  • a suitable APC e.g., a DC
  • an artificial composition mimicking an antigen-presenting cell for a period of time sufficient to activate said CTL in an antigen specific manner.
  • Engineered T cells may be used for adoptive T cell immunotherapy.
  • Autologous T cells may be engineered to express a defined T cell receptor (TCR) that are directed against target TAAs, either wild-type TCR, or mutated/engineered TCR towards a higher affinity to the antigen peptide/MHC molecule complexes.
  • TCR T cell receptor
  • a genetic engineered novel receptor consisting of a chimera between an antibody molecule and TCR segments (Chimeric Antigen Receptor, CAR) may be used for transduction of autologous T cells.
  • CAR Chimeric Antigen Receptor
  • CAR-engineered T cells combine TAA-recognized single-chain antibody with the activation motif of T cells, freeing antigen recognition from MHC restriction and thus breaking one of the barriers to more widespread application of ACL It means combining the high affinity of antibody to TAA with the killing mechanism of T cells. It had been bolstered that CAR-engineered T cells exhibited antitumor function to prostate cancer and other advanced malignancies.
  • CARs may include NKG2D based CARs (Sentman and Meetah, NKG2D CARs as cell therapy for cancer, 2014, Cancer I, 20(2): 156- 159); CD28z CARs and armored CARs ( reviewed by Pegram et al, CD28z CARs and armored CARs, 2014, Cancer J., 20(2): 127-133).
  • conjugates comprising active agents that can promote T cell migration and function may be transduced into engineered T cells to facilitate, after T cell infusion, the trafficking of infused T cells to tumor sites and penetrating the tumor microenvironments and functional maintenance.
  • ⁇ T cells are a special type of T lymphocytes which were found to act as interface for the cross talk between innate and cell-mediated immune cells, because of its expression of both natural killer receptors and ⁇ T cell receptors (Wu YL, et al. ⁇ T cells and their potential for immunotherapy. Int J Biol Sci 2014; 10: 119-35).
  • ⁇ TCR recognize non-peptide antigens like glycerolipids and other small molecules, polypeptides that are soluble or membrane anchored, and/or cross linked to major histocompatibility complex (MHC) molecules or MHC-like molecules in an antigen-independent manner (Reviewed by Bom et al, Diversity of gammadelta T-cell antigens. Cell Mol Immunol, 2013, 10(1): 13-20).
  • MHC major histocompatibility complex
  • ⁇ T cells have a unique role in the immune-surveillance against malignancies as they can directly recognize molecules that are expressed on cancer cells without need of antigen processing and
  • ⁇ T cells may be used to cross-present antigens to effector T cells with ⁇ receptor as effective APCs (See, e.g., US Pat. NO.: 8,338,173).
  • ⁇ T cells may be isolated and enriched in vitro from human peripheral blood cells. Isolated and expanded ⁇ T cells may be stimulated or loaded with tumor antigens comprised in conjugates, particles and formulations as discussed in the present application.
  • ⁇ T cells Prior to loading tumor antigens, in vitro isolated ⁇ T cells may be stimulated using (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP) or other small molecular weight non-peptide compounds with selectivity for ⁇ T cells, or other stimulators for induction of antigen-uptake, of presentation function and of expression of co-stimulatory molecules, e.g., phytohemagglutinin (PHA).
  • Stimulated ⁇ T cells may be loaded with one or more conjugates comprising one or more TAA peptide epitopes.
  • the loaded ⁇ T cells may be used to activate anti-tumor T cells as antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • TAA-loaded ⁇ T cells may be used to prime naive T cells to generate effector T cells.
  • compositions may be used as agonistic agents to ligate the positive costimulatory receptors, or blocking agents that attenuate signaling through inhibitory receptors.
  • a conjugate that comprises an antibody against the positive costimulatory receptor 4- IBB (CD 137), or an antibody against OX40, or antibodies against CD137 and OX40 may be used as an agonistic agent.
  • a conjugate that comprises an antibody against the inhibitory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4), or a fragment thereof may be used as a blocking agent to inhibit the immunosuppression.
  • CTL-4 inhibitory receptor cytotoxic T-lymphocyte antigen-4
  • agonistic agents and blocking agents of the present conjugates may be used in combination with other immunological conjugates, in particular, conjugates comprising active agents (e.g.TAAs, and antigenic peptides) that can stimulate initial antigen recognition of TCR.
  • active agents e.g.TAAs, and antigenic peptides
  • compositions such as conjugates, particles and/or formulations of the present invention may be used for cytokine based immunotherapy.
  • immunological conjugates of cytokines may be used to expand cytotoxic T cells. If a lower level of endogenous T cell priming has occurred in a tumor patient, cytokines, such as T cell growth factor, may be used to expand these activated T cells. In this strategy, conjugates comprising such cytokines, or particles/formulations that comprise such conjugates may be administered to the patient to expand activated T cells in vivo. For example, conjugates comprising IL-2, IL-7, IL-12 or in combination thereof, as a payload may be used for this purpose.
  • Conjugates comprising cytokines may also be used to induce killer cells (known as cytokine induced killer cells CIK) are a heterogeneous population of effector CD8+T cells with diverse TCR specificities, possessing non-MHC restricted cytolytic activities against tumor cells with the dual characteristics of T cells and NK cells, which could identify the target cells not only through the TCR and MHC, but also could through the Natural Killer (NK) cell activated receptor.
  • CIK cytokine induced killer cells
  • Conjugates and compositions of the present invention comprising antibodies or fragments thereof against a tumor specific antigen may be used for treatment of cancer.
  • Monoclonal antibodies that elicit an antigen-antibody response specific to tumor specific antigens (TAAs) induce various types of immune response including cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), etc. to attack cancer cells, thereby inducing cell death.
  • ADCC cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Antibodies against TAAs as target are disclosed in the art and can be incorporated to the conjugates or particles of the present invention, such as antibodies against 17-1 A (also known as EpCAM, EGP-40 or GA 733-2 (US Pat. No. :
  • AlloSCT Allogeneic stem cell transplantation
  • AlloSCT from a compatible donor peripheral blood has gained recognition as a potential immunotherapy for a number of different hematological malignancies and in advanced solid malignant tumors such as mRCC and castration resistant prostate cancer (CRPC).
  • Immunological conjugates of the present invention may be used to prime stem cells for transplantation.
  • conjugates and other compositions of the present invention may be used to enhance an innate immune response to increase the anti-cancer immunity in a subject.
  • conjugates of the present invention are used to block or reverse inhibitory mechanisms in tumors.
  • conjugates that comprise antibodies against PD-1, or antibodies against PD-L1, or both may be used to block the interaction between PD-1/PD-L1.
  • Another inhibitory receptor may be LAG-3 which is expressed on activated T cells.
  • IDO immunosuppressive enzyme indoleamine-2,3-dioxygenase
  • Blockade of IDO activity can be immune-potentiating in some tumors.
  • conjugates comprising one or more small molecule IDO inhibitor may be used to block its activity.
  • conjugates that comprise active agents which can deplete Treg cells, or myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment.
  • active agents may be antibodies against components of Treg cells MDSCs, for example, antiCD25 antibody.
  • an effective immunotherapy may combine different interventions including strategies to increase systemically the frequency of anti-cancer T cells, strategies to overcome distinct immune suppressive pathways within the tumor microenvironment and strategies to trigger innate immune activation and inflammation in tumor sites.
  • conjugates, particles and formulations comprising conjugates and vaccines may be used to treat cancer;
  • the cancer may be any cancer, including but not limited to any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Ho
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • An antigen or tumor antigen is prepared as a component of a conjugate.
  • the antigen or tumor antigen is a shared antigen or neoantigen.
  • the binding of conjugate moiety to antigen presenting cells is measured by flow cytometric analysis and/or fluorescence-activated cell sorting (FACS).
  • Antigen containing conjugate specific T cell lines are generated according to published methodologies.
  • Antigen containing conjugate stimulated PBMCs are cultured with T cells and evaluated using the ELISPOT assay (MBL, Nagoya, Japan) in 96-well ELISPOT plate (MultiScreen HTS, Millipore) and counted by an ELISPOT reader (CTL Technologies). Cytotoxicity
  • Antigen containing conjugate stimulated PBMCs are also tested for cytotoxicity against one or more cancer cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne l'immunothérapie anticancéreuse. L'invention concerne des conjugués et des nanoparticules comprenant des principes actifs qui peuvent provoquer une réponse immunitaire spécifique à un cancer. Lesdits conjugués comprennent une ou plusieurs fractions de ciblage qui sont reliées à ces principes actifs. L'invention concerne également des nanoparticules comprenant les conjugués de la présente invention pour améliorer l'administration de ces conjugués, et augmenter l'immunogénicité et diminuer la toxicité.
EP16833628.7A 2015-07-31 2016-07-29 Compositions et méthodes pour thérapies immuno-oncologiques Withdrawn EP3328377A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201562199414P 2015-07-31 2015-07-31
US201662332772P 2016-05-06 2016-05-06
PCT/US2016/044775 WO2017023779A1 (fr) 2015-07-31 2016-07-29 Compositions et méthodes pour thérapies immuno-oncologiques

Publications (2)

Publication Number Publication Date
EP3328377A1 true EP3328377A1 (fr) 2018-06-06
EP3328377A4 EP3328377A4 (fr) 2019-03-13

Family

ID=57943520

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16833628.7A Withdrawn EP3328377A4 (fr) 2015-07-31 2016-07-29 Compositions et méthodes pour thérapies immuno-oncologiques

Country Status (5)

Country Link
US (1) US20180221503A1 (fr)
EP (1) EP3328377A4 (fr)
AU (1) AU2016303497A1 (fr)
CA (1) CA2993429A1 (fr)
WO (1) WO2017023779A1 (fr)

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9517257B2 (en) 2010-08-10 2016-12-13 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US9850296B2 (en) 2010-08-10 2017-12-26 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
CA2807942C (fr) 2010-08-10 2021-07-27 Ecole Polytechnique Federale De Lausanne Agents therapeutiques se liant aux erythrocytes
SG11201504558TA (en) 2012-12-11 2015-07-30 Einstein Coll Med Methods for high throughput receptor:ligand identification
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
AU2015233084B2 (en) 2014-02-21 2020-12-17 Anokion Sa Glycotargeting therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US20170151281A1 (en) 2015-02-19 2017-06-01 Batu Biologics, Inc. Chimeric antigen receptor dendritic cell (car-dc) for treatment of cancer
US11319359B2 (en) 2015-04-17 2022-05-03 Alpine Immune Sciences, Inc. Immunomodulatory proteins with tunable affinities
AU2017250358B2 (en) 2016-04-15 2023-06-01 Alpine Immune Sciences, Inc. ICOS ligand variant immunomodulatory proteins and uses thereof
KR20190006495A (ko) 2016-04-15 2019-01-18 알파인 이뮨 사이언시즈, 인코포레이티드 Cd80 변이체 면역조절 단백질 및 그의 용도
JP2019522466A (ja) 2016-05-18 2019-08-15 アルバート アインシュタイン カレッジ オブ メディシン，インコーポレイティド 変異ｐｄ−ｌ１ポリペプチド、ｔ細胞調節多量体ポリペプチド、及びそれらの使用方法
WO2017201210A1 (fr) 2016-05-18 2017-11-23 Cue Biopharma, Inc. Polypeptides multimères modulateurs des lymphocytes t et leurs procédé d'utilisation
KR20230149857A (ko) 2016-07-07 2023-10-27 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 항체-애쥬번트 접합체
WO2018022945A1 (fr) 2016-07-28 2018-02-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd112 et utilisations associées
US11471488B2 (en) 2016-07-28 2022-10-18 Alpine Immune Sciences, Inc. CD155 variant immunomodulatory proteins and uses thereof
WO2018022957A1 (fr) * 2016-07-29 2018-02-01 Tarveda Therapeutics, Inc. Conjugués de liaison aux lymphocytes t et méthodes d'utilisation associées
CN116970061A (zh) 2016-12-22 2023-10-31 库尔生物制药有限公司 T细胞调节性多聚体多肽及其使用方法
EP3565829A4 (fr) 2017-01-09 2021-01-27 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs procédés d'utilisation
BR112019015797A2 (pt) 2017-02-01 2020-03-17 Modernatx, Inc. Composições de mrna terapêuticas imunomoduladoras que codificam peptídeos de mutação de oncogene de ativação
US20200010528A1 (en) 2017-03-15 2020-01-09 Cue Biopharma, Inc. Methods for modulating an immune response
WO2018232176A1 (fr) 2017-06-16 2018-12-20 The University Of Chicago Compositions et procédés d'induction d'une tolérance immunitaire
DK3661954T3 (da) 2017-08-03 2022-04-19 Amgen Inc Interleukin-21-muteiner og fremgangsmåder til behandling
WO2019033050A1 (fr) * 2017-08-11 2019-02-14 City Of Hope Molécule bispécifique de liaison à un antigène
KR20200085777A (ko) 2017-10-18 2020-07-15 알파인 이뮨 사이언시즈, 인코포레이티드 변이체 icos 리간드 면역조절 단백질 및 관련 조성물 및 방법
EP3737689A4 (fr) 2018-01-09 2021-12-01 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs procédés d'utilisation
KR20200110358A (ko) 2018-01-12 2020-09-23 암젠 인크 항-pd-1 항체 및 치료 방법
CN112040941A (zh) * 2018-03-15 2020-12-04 阿托莎医疗公司 诱导免疫应答的原位方法
EP3876977A1 (fr) 2018-11-06 2021-09-15 The Regents Of The University Of California Récepteurs antigéniques chimériques pour la phagocytose
EP3937984A1 (fr) 2019-03-15 2022-01-19 Bolt Biotherapeutics, Inc. Immunoconjugués ciblant le her2
US11013764B2 (en) 2019-04-30 2021-05-25 Myeloid Therapeutics, Inc. Engineered phagocytic receptor compositions and methods of use thereof
EP3980033A4 (fr) * 2019-06-05 2023-08-23 Emory University Photolyse pour déverrouiller des agents thérapeutiques de type protéines en cage
EP3986448A4 (fr) * 2019-06-19 2023-07-12 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs procédés d'utilisation
MX2022002613A (es) 2019-09-03 2022-06-02 Myeloid Therapeutics Inc Metodos y composiciones para la integracion del genoma.
US10980836B1 (en) 2019-12-11 2021-04-20 Myeloid Therapeutics, Inc. Therapeutic cell compositions and methods of manufacturing and use thereof
EP4106819A1 (fr) 2020-02-21 2022-12-28 Silverback Therapeutics, Inc. Conjugués d'anticorps de nectine-4 et leurs utilisations
WO2021231376A2 (fr) 2020-05-12 2021-11-18 Cue Biopharma, Inc. Polypeptides multimères modulateurs de lymphocytes t et leurs méthodes d'utilisation
WO2022006327A1 (fr) 2020-07-01 2022-01-06 Silverback Therapeutics, Inc. Conjugués d'anticorps anti-asgr1 et leurs utilisations
WO2022056014A1 (fr) 2020-09-09 2022-03-17 Cue Biopharma, Inc. Polypeptides multimères modulateurs des lymphocytes t du cmh de classe ii pour le traitement du diabète sucré de type 1 (dt1) et leurs procédés d'utilisation
WO2022098905A2 (fr) 2020-11-04 2022-05-12 Myeloid Therapeutics, Inc. Compositions de protéines de fusion chimériques modifiees et leurs procédés d'utilisation
WO2022259036A1 (fr) * 2021-06-08 2022-12-15 Beihaghi Maria Protéine multi-épitope à base de cytokine pour la liaison à des cellules positives de ccr7
CN114191410A (zh) * 2021-11-15 2022-03-18 深圳北航新兴产业技术研究院 一种诊疗一体化双亲微球缓释微球的制备方法
WO2023192992A2 (fr) * 2022-03-31 2023-10-05 The Wistar Institute Of Anatomy And Biology Anticorps bispécifiques engageant les cellules tueuses naturelles qui ciblent siglec-7
WO2023215848A1 (fr) * 2022-05-06 2023-11-09 Umoja Biopharma, Inc. Particule virale à molecules de stimulation de surface
CN115590947A (zh) * 2022-05-27 2023-01-13 苏州大学（Cn） 联合免疫用药物、温敏凝胶组合物、趋化因子颗粒及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1897548B2 (fr) * 2003-02-28 2024-05-22 The Johns Hopkins University Régulation des lymphocytes T
ES2546509T3 (es) * 2009-06-25 2015-09-24 Commissariat à l'énergie atomique et aux énergies alternatives Polipéptidos multiméricos de HLA-G que incluyen monómeros alfa1-alfa3 y usos farmacéuticos de los mismos
MY172519A (en) * 2012-12-28 2019-11-28 Tarveda Therapeutics Inc Solid polymeric controlled release nanoparticle
CA3185368A1 (fr) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Generation efficace de lymphocytes t ciblant une tumeur derives de cellules souches pluripotentes

Also Published As

Publication number Publication date
CA2993429A1 (fr) 2017-02-09
US20180221503A1 (en) 2018-08-09
EP3328377A4 (fr) 2019-03-13
WO2017023779A1 (fr) 2017-02-09
AU2016303497A1 (en) 2018-03-01

Similar Documents

Publication Publication Date Title
EP3328377A1 (fr) Compositions et méthodes pour thérapies immuno-oncologiques
CA2993478A1 (fr) Compositions et methodes d'immunomodulation
Song et al. Nanomaterials for cancer immunotherapy
ES2952421T3 (es) Refuerzo del efecto de las células T modificadas por CAR por medio de vacunación con ácido nucleico
JP2021523110A (ja) 遺伝子発現のためのナノ粒子及びその使用
JP2019515888A (ja) 細胞免疫療法前の細胞毒性プレコンディショニングの代替
KR20190039597A (ko) 세포 치료요법을 위한 항상성 활성 사이토킨 수용체
US11771718B2 (en) Polypeptide compositions comprising spacers
Hong et al. Addressing barriers to effective cancer immunotherapy with nanotechnology: achievements, challenges, and roadmap to the next generation of nanoimmunotherapeutics
ES2342606T3 (es) Blanco in vivo de celulas dentriticas.
JP7295360B2 (ja) 固形腫瘍細胞及びエスケープバリアントを治療するための足場
ES2924138T3 (es) Nueva composición farmacéutica que comprende partículas que comprenden un complejo de un polirribonucleótido de doble cadena y una polialquilenimina
Park et al. Personalized combination nano-immunotherapy for robust induction and tumor infiltration of CD8+ T cells
WO2020252404A1 (fr) Compositions de ca2 et procédés de régulation accordable
CA3108610A1 (fr) Presentation de peptides a des cellules presentatrices d'antigene a l'aide d'un vehicule lipidique
WO2018022957A1 (fr) Conjugués de liaison aux lymphocytes t et méthodes d'utilisation associées
WO2022250811A2 (fr) Nanoparticules pour la programmation de cellules spécifiques d'un antigène et leurs utilisations
EP4251198A1 (fr) Combinaisons de conjugués de médicament à petites molécules et de lymphocytes cytotoxiques exprimant un récepteur antigénique chimérique et méthodes de traitement du cancer à l'aide de celles-ci
US20240139241A1 (en) Compositions and methods for conditioning patients for cell therapy
Bogacz et al. Modern immunotherapy using CAR-T cells in haemato-oncology and solid tumors
JP2023518935A (ja) 抗原特異的ｔ細胞受容体およびｔ細胞エピトープ
WO2024092028A2 (fr) Régimes de traitement combinés pour le traitement du cancer
TW202305119A (zh) 用於產生抗原特異性ｔ細胞及治療疾病之材料及方法
WO2022034523A1 (fr) Composés d'induction de phagocytose et leurs procédés d'utilisation
Zheng Arming adoptively transferred T-cells with drug-loaded nanoparticles for cancer immunotherapy

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20180216

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20190212

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 31/215 20060101AFI20190206BHEP

Ipc: C07K 2/00 20060101ALI20190206BHEP

Ipc: A61K 31/337 20060101ALI20190206BHEP

Ipc: A61K 31/282 20060101ALI20190206BHEP

17Q First examination report despatched

Effective date: 20200403

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20200930